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10085294 | Cells were grown in yeast extract peptone dextrose (YPD) or in synthetic complete (SC) media as previously described . For experiments in which cells were induced with mating pheromone, synthetic α-factor (Department of Molecular Biology, Syn/Seq facility, Princeton University) was added to a final concentration of 10 μg/ml. For experiments localizing GFP-Kar9p, cultures of each strain containing the plasmid pMR3465 were grown to saturation in SC media without leucine (SC-leucine). The precultures were then diluted 500-fold into SC-leucine media containing 2% raffinose instead of glucose and grown for 15–18 h at 30°C to early exponential phase. Expression of GFP-Kar9p was induced for 2–2.5 h at 30°C by the addition of galactose to 2%. To express GFP-Kar9p in shmoos, α-factor was added at the time of galactose induction. Cells were fixed by the addition of formaldehyde to 3.7% for 10 min and washed twice with PBS. For localization of GFP-Bni1p, cells were treated as described above, with the exception that they were grown in SC media without uracil and the expression of GFP-Bni1p was induced by the addition of galactose for 4 h. For determination of the cell-cycle distribution and nuclear position in different mutants, saturated overnight cultures were subcultured into YPD preincubated at either 30° or 14°C. Cells were grown to mid-exponential phase and fixed in methanol:acetic acid (3:1), washed twice in PBS, and stained with 4′,6-diamidino-2-phenylindole (DAPI; Accurate Chemical and Scientific Corp.). For the analysis of the budding pattern, a/α diploid cells homozygous for the indicated mutations were grown to late exponential phase in YPD at 30°C. Cells were then stained with Calcofluor white ( Sigma Chemical Co. ) to visualize bud scars as described . Only those cells with three or more bud scars were scored. If the bud scars were located at both poles, the cells were scored as bipolar. All other patterns were scored as random. The wild-type strain, MS1556, expressing the GFP-Kar9p plasmid was grown to early exponential phase as described above. Mitotic cells or pheromone-treated cells (shmoos) were concentrated 10-fold, and latrunculin-A (LAT-A) was added to 200 μm (Molecular Probes, Inc.) in DMSO for 10 min. Control cells were treated with an equivalent volume of DMSO. Cells were then collected by centrifugation and fixed for observation of the GFP fluorescence as described above or for staining of actin with rhodamine-phalloidin. For rhodamine-phalloidin staining, cells were collected by centrifugation and resuspended in 0.1% Triton X-100, 3.7% formaldehyde in PBS at room temperature for 30 min. Cells were washed once in PBS and resuspended in 3.7% formaldehyde/PBS at 30°C for 2 h. Cells were washed twice in PBS and resuspended in 90 μl PBS. Cells were stained by the addition of 30 μl rhodamine-phalloidin (0.2 U/μl; Molecular Probes, Inc.) in methanol at room temperature in the dark for 90–120 min. Cells were then washed once in PBS and scored by fluorescence microscopy. Yeast strains used in the course of this study are listed in Table I . Plasmids used in strain construction are listed in Table II . Isogenic deletion mutants were constructed in the wild-type strain, MS1556, derived from S288C. All oligonucleotides used for deletion construction were made by the Department of Molecular Biology Syn/Seq Facility, Princeton University. The bni1 Δ was made by cutting p321 with HindIII and XhoI and transforming into MS1556. This resulted in the bni1 Δ strains, MS5212 and MS5340. To construct bnr1 Δ:: HIS3 , all but the first codon of BNR1 was replaced with HIS3 using the one-step gene replacement method . The disruption fragment was generated by PCR using the following two oligonucleotides ( BNR1 sequence is shown in normal font and HIS3 sequence is shown in italics): 5′ TTTTGAAGATTACATAGTGATGAT GATCGTGACACAAAAGCAGATAAAAAAATAGCACAATCATCAGCGATG CCGTTTTAAGAGCTTGGTG 3′ and 5′ AGCGATTGCGAATATTGTCCATTTCTTTATATAAGCTCCACAACTACATAAATACTAAGTC T T CACTA CGAG TTCAAGAGAAAAAAAAAG 3′ . Plasmid pRS403 was used as the template for the HIS3 portion of the construct. MS1556 was transformed to HIS3 prototrophy to create MS5794. Replacement of BNR1 was confirmed by PCR analysis. To construct bud6 Δ:: HIS3 , all but the first codon of BUD6 wasreplaced by the HIS3 gene using the one step gene replacement method . The disruption fragment was generated by PCR using the following forward and reverse primers ( BUD6 sequence is in normal font and HIS3 sequence is shown in italics): 5′ ACCCAAGAAAAG- GGAAAAAGAGTAGAAGCTGGCTGCCAAATTGGTGTAGTAATCCT CGTATTATTTTAAAATTAGATG CCG TTTT AAGAGC TT G-GTG 3′ and 5′ TATTGCCGCAAACTTTGTAATAAGCCAAAAG-CACTAA T C T C TTTTCCGTTAGC TTTCATAAAATTAGTGTATTTA CGAGTTCAAGAGAAAAAAAAAG 3′. Plasmid, pRS403 , was used as the template for the HIS3 portion of the disruption construct. MS1556 was transformed to yield the BUD6 deletion strain, MS5849 and MS5850. The deletion was confirmed at both the 5′ and 3′ ends by PCR analysis. To construct a pea2 Δ mutation, pNV44 was digested with KpnI and BamH1. The resulting 2.0-kb band was gel purified and transformed into the wild-type strain, MS1556, resulting in disruption of the endogenous PEA2 locus by the one-step gene replacement . Two independent colonies, MS5229 and MS5230, were isolated. A spa2 Δ mutation was made by the one-step gene replacement method. p210 (in which the SacI/SphI fragment of SPA2 was replaced by URA3 ), a kind gift from Michael Snyder (Yale University, New Haven, CT), was digested with HindIII and SalI. A 4.0-kb band was gel purified and transformed into the wild-type strain, MS1556, creating the SPA2 disruptants, MS5208 and MS5209. Microscopy was carried out on an Axiophot microscope equipped with a 1.4 NA 100× Neofluor lens ( Carl Zeiss, Inc. ) or a 1.3 NA 100× UplanFl iris lens ( Olympus Corp. ). Images were recorded using a SIT Video Camera 3200 with a camera controller C2400 (Hamamatsu Corp.). Images were initially processed using Omnex Image processing unit (Imagen) and captured to computer disk using a Scion image capture board. Adobe Photoshop 4.0 was used to further optimize contrast. Kar9p is a cortical protein that plays an important role in orienting cytoplasmic microtubules. To understand the mechanism of Kar9p microtubule orientation, we sought to identify the cellular components involved in localizing Kar9p to its cortical site. Previous work demonstrated that Kar9p's cortical localization is independent of microtubules . Therefore, Kar9p's cortical localization must be dependent on other peripheral determinants. As a first step, we used rhodamine-phalloidin to determine whether GFP-Kar9p colocalizes with cortical actin in pheromone-treated cells. In every case ( n = 20), we found GFP-Kar9p coincident with actin at the tip of the shmoo (data not shown). Although in several cases we observed a well-resolved actin dot that coincided with the GFP-Kar9p, most often the cortical actin dots were too concentrated to resolve individual structures. These results suggested that Kar9p might be associated with cortical actin structures. To determine whether the actin cytoskeleton is required for Kar9p localization, we used the actin-depolymerizing drug LAT-A. Confirming earlier work , we found that the actin network was 100% depolymerized within 10 min after treatment with LAT-A, as shown by staining with rhodamine-phalloidin (data not shown). We first examined the localization of a functional GFP-Kar9p construct in pheromone-treated cells. In the control experiments, 84% of shmoos exhibited a single dot of GFP-Kar9p fluorescence at the tip of the mating projection . In contrast, when shmoos were treated with LAT-A, only 8% of the shmoos exhibited the cortical dot at the tip of the projection. Instead, in 74% of the LAT-A–treated shmoos, GFP-Kar9p fluorescence was found in a line extending from the nucleus. Based on previous experiments, we expect that this corresponds to cytoplasmic microtubules . In addition, 19% of the LAT-A–treated shmoos exhibited no localization of the GFP-Kar9p fluorescence. Thus, depolymerization of the actin cytoskeleton results in rapid loss of the cortical localization of Kar9p and redistribution onto the cytoplasmic microtubules. We next examined the requirement for polymerized actin for Kar9p localization in mitotically dividing cells. Unbudded and most post-anaphase large budded cells do not exhibit localized GFP-Kar9p , so we restricted our observations to cells with medium to large buds. In the control cells, 72% displayed a single dot of GFP-Kar9p fluorescence at the tip of the bud . When mitotic cells were treated with LAT-A, only 13% exhibited the dot at the tip of the bid. Instead, 59% of the cells exhibited a dot of GFP-Kar9p fluorescence at two alternate locations, coincident with the DAPI staining and/ or close to the mother-bud neck . The GFP fluorescence coincident with the nuclear DAPI stain is likely to correspond to SPB localization. Another 21% of the LAT-A–treated mitotic cells exhibited no localized GFP-Kar9p fluorescence. Thus, in >80% of LAT-A–treated cells, GFP-Kar9p localization was not found at the tip of the bud. From these results, we conclude that GFP-Kar9p localization in both shmoos and mitotically dividing cells is dependent on polymerized actin. Although Kar9p's localization was actin dependent, it was found only as a single dot and therefore did not colocalize with other cortical actin patches at the shmoo tip and bud cortex. Therefore, we reasoned that additional cortical actin-associated proteins might restrict Kar9p localization to one site. In this case, mutations in a subset of specific actin-associated proteins would eliminate or alter Kar9p localization. To test this idea, we used pheromone-treated cells to assay GFP-Kar9p localization in mutants that affect actin, polarization, and/or mating. Most mutations had no effect on GFP-Kar9p localization to the tip of the shmoo projection. These included genes affecting actin function, ABP1 and SLA1 ; genes involved in GTPase signaling, BOI2 , RSR1/BUD1 , RGA1 , and MSB1 ; and genes affecting cell fusion, RVS161 , FUS1 , and FUS2 . Furthermore, GFP-Kar9p localization in mitotic cells was not affected by mutations in the genes encoding two microtubule-dependent motor proteins, KIP3 and DHC1 . However, GFP-Kar9p localization in shmoos was affected by four mutations implicated in actin organization and polarization, spa2 , pea2 , bud6 , and bni1 . SPA2 and PEA2 are required for polarization in mating cells and mutations result in broader peanut-shaped shmoo projections . In mitotic cells, SPA2 and PEA2 are required for the diploid-specific bipolar budding pattern. Spa2p and Pea2p physically interact with each other and are interdependent for their own localization to the tips of shmoo projections . In the wild-type strain, 86% of the cells displayed GFP-Kar9p localization as a single dot at the tip of the shmoo projection. In contrast, only 15% of spa2 Δ and 11% of pea2 Δ shmoos exhibited GFP-Kar9p fluorescence as a single dot. Instead, 50% of spa2 Δ and 55% of pea2 Δ shmoos showed a new pattern in which GFP-Kar9p was found in a broad region over the shmoo tip. In addition, both mutants showed a significant number of cells with GFP-Kar9p in a line or in multiple “speckles” . BUD6 is also required for the diploid-specific bipolar budding pattern and Bud6p (Aip3p) has been shown to interact with actin . In the bud6 Δ strain, only 41% of shmoos exhibited Kar9p fluorescence as a single dot . Instead, a large percentage of the bud6 Δ shmoos exhibited GFP-Kar9p fluorescence as a line . BNI1 encodes a formin homologue that has been implicated in the regulation of the actin cytoskeleton and cell polarization , as well as bipolar budding . Bni1-GFP also localizes to the shmoo tip . When GFP-Kar9p localization was examined in bni1 Δ shmoos, only 12% exhibited Kar9p fluorescence as a cortical dot. Instead, 37% of the bni1 Δ shmoos exhibited GFP-Kar9p in a line pattern and 41% in a speckled pattern throughout the shmoo . Because Kar9p also functions to orient cytoplasmic microtubules during vegetative growth , we sought to determine whether these four mutants also affected Kar9p localization during mitosis. In 85% of wild-type medium-to-large budded cells, GFP-Kar9p localized to a single dot at the tip of the bud. In the spa2 Δ and pea2 Δ cells, there was a modest reduction in the frequency of GFP-Kar9p localization to the bud tip, down to 75 and 74%, respectively . Although modest, the reduction was observed in three independent experiments. Only 38% of the bud6 Δ cells had a dot of GFP-Kar9p fluorescence at the tip of the bud. In 41% of the bud6 Δ cells, GFP-Kar9p was mislocalized to the mother-bud neck and/or a region coincident with DAPI staining. Finally, in the bni1 Δ cells, only 14% of cells had a dot of GFP-Kar9p at the tip of the bud. Instead, 70% of these cells had Kar9p fluorescence at the mother-bud neck and/ or a region coincident with DAPI staining . Therefore, all four mutations affected Kar9p localization, although only in the bni1 Δ strain was the magnitude similar to that of actin depolymerization. Because the deletion of BNI1 had such a strong effect on Kar9p localization, we next determined whether the two proteins are interdependent for localization. Accordingly, we examined the localization of Bni1p using a fully functional GFP-Bni1p fusion (a generous gift from Charlie Boone, Queens University, Kingston, Canada) in kar9 Δ mutant shmoos and mitotic cells. In comparing kar9 Δ to wild type, there were no obvious differences in the localization pattern of GFP-Bni1p to the presumptive bud site or the tip of the growing bud (data not shown). Therefore, Bni1p's association with the cortex is not dependent on Kar9p. BNI1 -related ( BNR1 ) is another yeast gene encoding a formin homology domain protein. Bnr1p shares some functions with Bni1p . We therefore determined whether BNR1 was also required for GFP-Kar9p localization. In both shmoos and mitotic cells, GFP-Kar9p localization in an isogenic bnr1 Δ strain was indistinguishable from wild type (data not shown). Thus, localization of Kar9p to the cortex is a specific function of Bni1p. Since spa2 Δ, pea2 Δ, bud6 Δ, and bni1 Δ mutants resulted in an altered localization for GFP-Kar9p, we wanted to determine whether these mutants also exhibited defects in nuclear migration like kar9 Δ. Nuclear position was scored in asynchronous cultures using the DNA-specific fluorescent dye, DAPI (Table III ). In the wild-type control, 20% of the cultures were large budded and, of these, 96% showed normal nuclear positions (i.e., located at the bud neck, or in anaphase across the bud neck, or in telophase with one nucleus each in the mother and bud). In contrast, 40% of kar9 Δ mutants were large-budded (Table III ), suggesting that kar9 Δ has a cell cycle delay in mitosis. Consistent with our previous report , nuclear positioning was aberrant in up to 50% of the kar9 Δ large-budded cells (i.e., nuclei not located at the bud neck, or anaphase or telophase occurring in the mother cell). The spa2 Δ, pea2 Δ, and bud6 Δ mutants exhibited no obvious defects in nuclear position within the large-budded cells. However, like kar9 Δ, the bni1 Δ strain exhibited a significant defect in nuclear position; in 23% of the large-budded cells, anaphase was occurring within the mother cell. In contrast, only 2% of wild-type large-budded cells exhibited this phenotype (Table III ). Unlike kar9 Δ, only 5% of bni1 Δ large-budded cells contained two nuclei within the mother cell (compared with 18% in kar9 Δ). Similar results were obtained when the spa2 Δ, pea2 Δ, bud6 Δ, and bni1 Δ cultures were grown at 14°C (data not shown). Therefore, we conclude that bni1 Δ is a nuclear migration mutant. However, the nuclear migration defect of bni1 Δ is not as severe as that of kar9 Δ. We next examined nuclear position in cells treated with mating pheromone. In wild type, the nuclei migrated to the base of the shmoo projection in 66% of the cells. As previously reported , the kar9 Δ mutants exhibited a severe defect in nuclear migration to the shmoo projection, with 71% of the nuclei found in the center of the shmoo body (Table IV ). The spa2 Δ and pea2 Δ mutants did not exhibit a nuclear migration defect, and may have localized better than the wild type, consistent with the continued localization of Kar9p at the shmoo tip. However, in 58% of the bud6 Δ shmoos and 72% of the bni1 Δ shmoos, the nuclei failed to migrate into the shmoo projection and were instead positioned in the center of the shmoo body (Table IV ). Therefore, during mating, bud6 Δ mutants exhibited a moderate defect and bni1 Δ mutants exhibited a major defect in nuclear positioning. We next examined the microtubule morphologies of the mutants. In >90% of wild-type shmoos, the cytoplasmic microtubules were present as a single bundle that extended from the nucleus to the tip of the shmoo projection . As previously reported, only 15% of the kar9 Δ shmoos contained cytoplasmic microtubules that extended to the shmoo tip. In most kar9 Δ shmoos, the cytoplasmic microtubule bundle was misoriented and extended in random directions. In the spa2 Δ mutant shmoos, only 52% showed a single bundle of microtubules oriented towards the shmoo tip. Instead, 29% of the spa2 Δ mutant shmoos exhibited a “spray” of microtubules extending to the shmoo tip, and 10% showed supernumerary microtubules oriented away from the shmoo tip . The pea2 Δ mutant showed a similar pattern, albeit significantly less severe than that of spa2 Δ. The presence of additional cytoplasmic microtubules making contact with the cortex is consistent with Kar9p localizing over a broader region of the shmoo tip. The bni1 Δ strain also showed a significant defect in microtubule orientation in the shmoo. Like spa2 Δ, only 50% of bni1 Δ shmoos exhibited the normal microtubule pattern. However, unlike spa2 Δ, 39% of the bni1 Δ mutant shmoos exhibited misoriented microtubules similar to the pattern seen with the kar9 Δ . Thus, in mating cells, the defect in microtubule orientation in the bni1 Δ mutants is consistent with the aberrant localization of Kar9p. We next examined the microtubule morphology in bni1 Δ mitotic cells. For this purpose, we focused on those cells exhibiting a nuclear positioning defect such that anaphase was occurring within the mother cell. As reported previously , when the rare wild-type cells with anaphase in the mother were examined, the cytoplasmic microtubules nearly always extended into the bud (90%, Table V ). In contrast, in the kar9 Δ mutant, the cytoplasmic microtubule bundles infrequently extended into the bud (31%) and were more often found in the mother cell (48%). However, in 79% of the bni1 Δ mutant, the cytoplasmic microtubules were correctly extended into the bud. This result suggests that either the bni1 Δ mutation also suppresses the microtubule orientation defect created by mislocalized Kar9p or that enough Kar9p was correctly localized in the bni1 Δ mutant to facilitate microtubule orientation. To differentiate between these two models, we created kar9 Δ bni1 Δ double mutants and examined them for defects in nuclear positioning and cytoplasmic microtubule orientation. In 49% of the kar9 Δ bni1 Δ double mutant cells, cytoplasmic microtubules were misoriented (Table V ) like the kar9 Δ mutant. Therefore, Kar9p was still responsible for the normal microtubule orientation found in the bni1 Δ single mutant cells, in spite of the defect in Kar9p localization. These results suggest that Kar9p localization must be partially independent of Bni1p. This result would also be consistent with the weaker defect in nuclear migration seen for bni1 Δ compared with kar9 Δ. We next examined the double mutants for defects in nuclear positioning. Compared with the kar9 Δ single mutant, the kar9 Δ bni1 Δ double mutant large-budded cells were slightly more severe for defects in nuclear positioning (47 vs. 33% aberrant, Table VI ). In unbudded cells, however, a significantly more severe defect was observed. In unbudded cells, very low percentages of abnormal cells were found in wild-type, kar9 Δ, and bni1 Δ single mutants (0, 4, and 0.5%, respectively). However, in the kar9 Δ bni1 Δ double mutant, 22% of the cells were either multinucleate or anucleate. Therefore, the double mutant cells often appeared to progress through cytokinesis, which almost never occurred with the single mutants. Because of the synthetic lethality between kar9 Δ and dhc1 Δ, Kar9p and dynein are thought to function in separate, partially redundant pathways for nuclear migration . Because spa2 Δ, pea2 Δ, bud6 Δ, and bni1 Δ mutants each altered GFP-Kar9p localization, we wanted to know whether any of these mutations would show genetic interactions similar to those observed with kar9 Δ. Isogenic marked deletions of each gene were created and crossed to marked kar9 and dhc1 deletions. The viability of the double mutants was assayed for growth 2–3 d after spore germination. The results of the crosses are summarized in Table VII . When spa2 Δ, pea2 Δ, bud6 Δ, or bni1 Δ were crossed with kar9 Δ, all four double-mutant combinations were viable (Table VII , 1–4). Similarly, the spa2 Δ dhc1 Δ, and pea2 Δ dhc1 Δ double mutants were viable (Table VII , 6–8). The bud6 Δ dhc1 Δ double mutants were viable, but exibited a mild growth defect (Table VII , 8). However, when bni1 Δ was crossed to dhc1 Δ, all the predicted double mutants were barely viable and formed microcolonies (Table VII , 9). Similar microcolonies were also observed in synthetic lethal genetic interactions seen with kar9 Δ, including dhc1 Δ, bik1 Δ, and kip2 Δ . Therefore, we consider this to be a synthetic lethal interaction and conclude that Bni1p acts in the Kar9p pathway for nuclear migration. As a control, crosses were performed using a deletion of the related formin homology domain protein, Bnr1p. The bnr1 Δ mutation was not synthetically lethal in combination with kar9 Δ or dhc1 Δ (Table VII , 5 and 10). Therefore, the interaction seen between dhc1 Δ and bni1 Δ is specific to Bni1p and not a general feature of formin homology proteins. In previous work, Kip3p was shown to function in the KAR9 pathway for nuclear migration . Because Bni1p also appears to function in the KAR9 pathway, we crossed the bni1 Δ to the kip3 Δ to determine whether Bni1p and Kip3p might also function in a common pathway. We found that the bni1 Δ kip3 Δ double mutant is viable, as was also reported by Lee et al. . This suggests that Kar9p, Kip3p, and Bni1p function in a common pathway. Accordingly, the kar9 Δ bni1 Δ kip3 Δ triple mutant would also be predicted to be viable. We found that the kar9 Δ bni1 Δ kip3 Δ triple mutant was indeed viable (Table VII , 12), supporting the hypothesis that Kar9p, Kip3p, and Bni1p function together in a common pathway. The four mutations that affect Kar9p localization, spa2 Δ, pea2 Δ, bud6 Δ, and bni1 Δ, share an additional common phenotype of being defective in the diploid-specific bipolar budding pattern . This provided an additional way of addressing whether Kar9p is simply a Bni1p-associated protein or functions specifically for microtubule orientation. Therefore, we examined whether kar9 Δ mutants are also defective for bipolar budding. Diploid cells with three or more bud scars were scored to determine whether they showed the bipolar or random budding pattern (see Materials and Methods). In the wild-type control strain, 95% of the cells exhibited a bipolar budding pattern and 5% showed a random pattern ( n = 263). In the pea2 Δ strain, only 28% of the cells exhibited the bipolar budding pattern, while 72% budded randomly ( n = 201). Unlike the pea2 Δ mutant, the kar9 Δ mutant exhibited the wild-type budding pattern (90% bipolar, n = 300). The kar9 Δ mutants also did not display a defect in the haploid-specific axial budding pattern (data not shown). Taken together with the observation that kar9 Δ mutant shmoos do not exhibit polarization defects, and that Bni1p localizes normally in the kar9 Δ mutants, these results argue strongly that Kar9p's function with Bni1p is specific to the nuclear migration pathway. In yeast, both actin and the cytoplasmic microtubules have been implicated in normal spindle orientation and movement, suggesting a possible interaction between these two cytoskeletal networks . However, the mechanism by which the actin cytoskeleton might contribute to spindle orientation has not been clear. Studies on centrosomal rotation in C . elegans and nuclear movement in yeast have also shown the involvement of cortical factors in microtubule positioning and orientation. However, to date, only Kar9p has been identified as a likely candidate for one of the cortical proteins required for cytoplasmic microtubule orientation. In this study, we demonstrated that Kar9p localization is dependent upon the actin cytoskeleton. We have also shown that Kar9p localization is strongly influenced by Bni1p, a formin involved in actin cytoskeletal functions, polarization, budding, and cell fusion processes. Like Kar9p, Bni1p was found to participate in nuclear migration processes during both shmooing and mitosis. Genetic analysis suggests that Bni1p functions in the Kar9p pathway rather than the dynein pathway for nuclear migration. Taken together, these data suggest that Kar9p and Bni1p functionally link the actin and microtubule networks to promote proper spindle orientation and nuclear migration. Three other proteins were found to influence Kar9p localization in mating cells, Bud6p, Spa2p, and Pea2p. Spa2p and Pea2p are required for polarization in mating cells and in their absence the shmoo tip is broader. In these two mutants Kar9p localization was spread over a large area of the shmoo tip. Thus, it seems likely that Pea2p and Spa2p and other proteins help to cluster Kar9p into a single focus at the shmoo tip. Taken together, these observations suggest a model for nuclear orientation in mitotic and mating cells. As the nascent bud emerges, actin patches become associated with the bud cortex. Similarly, actin patches associate with the cortex at the tip of the mating projection. One or a subset of actin patches becomes integrated into a specialized site that we will refer to as the “polarization complex,” normally at or near the tip of the bud and the mating projection. Additional actin-associated proteins playing roles in polarized growth would be recruited to the specialized polarization complex. Among these would be Bni1p, Spa2p, and Pea2p. Each of these proteins would interact with a subset of other components in the complex and each may have cortical associations that are independent of actin. Some of the proteins, perhaps including Bni1p, may play key roles in cross-linking multiple proteins and thereby play a major “scaffolding” role. Loss of such proteins would affect the localization of several proteins to the complex. The specific proteins required might also differ between mating and mitosis. Finally, other proteins, such as Kar9p, would be recruited to more peripheral locations on the polarization complex. Such peripheral proteins may serve as “docking sites” or “adapters” for extrinsic elements such as the microtubules that need to identify and interact with the cortical polarization complex. Thus, the assembly and maintenance of the actin-associated polarization complex provides a unique molecular “address” within the cell that can be “read” by different cellular systems through specific adapter molecules. We have identified Bni1p as being required for nuclear migration in mating and mitosis because of its role in Kar9p localization. In pheromone-treated cells, the bni1 Δ mutant exhibits a strong defect in microtubule orientation, consistent with the defect in Kar9p localization. The role of Bni1p in mitotic cells is more complex. Although Kar9p was mislocalized away from the bud tip in mitotic bni1 Δ mutant cells, the overall effect was less dramatic. In the mitotic cells, Kar9p localization was shifted to secondary sites at the SPB and/or bud site and the cytoplasmic microtubules were not grossly misoriented by the onset of anaphase. Several explanations for this are possible. First, some 14% of the bni1 Δ cells showed detectable Kar9p at the tip of the bud. It is possible that different levels of Kar9p at the bud tip are required for microtubule orientation and subsequent nuclear migration. Perhaps there is enough Kar9p at the bud tip to allow correct microtubule alignment, but not enough Kar9p to activate some other component necessary for nuclear migration, such as Kip3p. Second, although Kar9p was not localized to the bud tip in the mutant, it was localized near the bud neck. Microtubules contacting Kar9p at the bud neck would be properly oriented to eventually contact the bud cortex after continued assembly. This would be consistent with the analysis of the double mutant that indicated that in the bni1 Δ mutant the orientation of the cytoplasmic microtubules was still dependent on Kar9p. The residual localization of GFP-Kar9p in the bni1 Δ, the dependence of microtubule orientation on Kar9p in the bni1 Δ, and the fact that the kar9 Δ mutant exhibits a more severe nuclear migration defect than the bni1 Δ mutant suggests that Kar9p is at least partially active in the absence of Bni1p. Taken together, the simplest explanation for these observations is that Kar9p has substantial residual localization to the cortex, independent of Bni1p. In the absence of Bni1p, the level of GFP-Kar9p at the cortex may simply fall below the limit of detection in most cells. Nevertheless, the reduction in Kar9p localization would be sufficient to compromise the Kar9p pathway for nuclear migration. One prediction of these results is that Kar9p localization would be partially dependent on additional cortical proteins. Independently, Lee et al. have also identified Bni1p as being required for nuclear migration. In their work, dynamic studies of nuclear migration and microtubule dynamics clearly show a defect in cytoplasmic microtubule function. In addition, they also find that Kip3 and Bni1p are likely to be part of the same nuclear migration pathway. In contrast to bni1 Δ, there was no obvious defect in nuclear migration in the spa2 Δ, pea2 Δ, and bud6 Δ cells. This is not surprising given their milder defects in Kar9p localization. Our results showing that bud6 Δ does not exhibit a significant nuclear migration defect are in contrast to results previously reported . This may be due to differences in strain background or partially toxic effects of the disruption mutant used in that study. In support of this, we find that the bud6 Δ:: URA3 strain did exhibit a synthetic phenotype and temperature sensitivity in combination with dhc1 Δ. Although kar9 and bni1 mutants exhibit several similarities and genetically lie in the same pathway for nuclear migration, several lines of evidence suggest that they also have distinct functions. First, unlike bni1 Δ mutants , kar9 Δ mutants do not exhibit defects in actin localization . Second, kar9 Δ mutants do not exhibit defects in the bipolar budding pattern. Third, unlike bni1 Δ mutants , kar9 Δ did not form round-shaped shmoos or show other obvious defects in polarization. Fourth, unlike bni1 Δ mutants, kar9 Δ mutants do not exhibit a severe mating defect . Fifth, although Bni1p is required for efficient Kar9p localization, Kar9p is not required for GFP-Bni1p localization in mitotically dividing cells or shmoos. Thus, Bni1p mediates a series of functions in mating and mitosis that are not part of Kar9p's repertoire of functions. If Bni1p's only function in nuclear migration were to localize Kar9p, then the bni1 Δ kar9 ΔΔ double mutant should not be more severe than the kar9 Δ single mutant. However, the double mutant has a more severe phenotype than either single mutant alone, as evidenced both by a slight increase in the number of aberrant large-budded cells and by an increase in the percentage of multinucleate and anucleate unbudded cells (Table VI ). Thus, Bni1p may have a secondary role in nuclear migration in addition to the localization of Kar9p. However, if Bni1p does have a secondary role independent of Kar9p, its contribution to nuclear migration must be relatively minor. Otherwise, the additive effects of the two independent defects should lead to a severe growth defect for the double mutant. Instead, we find that the double mutant grows no worse than the single mutants, identical to the wild type. That anucleate cells are observed in the bni1 Δ kar9 Δ double mutant combination raises the additional possibility that Kar9p and/or Bni1p may play an inhibitory role in cytokinesis. This might be true if one or the other is part of the checkpoint mechanism that coordinates nuclear migration and cytokinesis. In addition to bud tip localization, both Bni1p and Kar9p are also localized at the mother-bud neck in a minor fraction of large budded cells . More likely, the increased severity of the double mutant may cause some cells to eventually complete cytokinesis without proper nuclear migration. Consistent with the observation that they all contribute to polarization, Spa2p, Pea2p, and Bud6p have been isolated as a stable complex . Furthermore, Spa2p interacts with Bni1p by two-hybrid analysis . Yet mutations in these proteins affected Kar9p localization differently, with only mild mitotic defects associated with spa2 Δ and pea2 Δ, and none apparent for bud6 Δ. In shmoos and mitotic cells, the most severe effect on Kar9p localization occurred in the bni1 Δ mutants. Nevertheless, all four of these mutants affect polarization in a similar manner and to a similar degree. Thus, the effects of bni1 Δ on Kar9p localization are not likely to be due to general defects in polarization. All four mutants, spa2 Δ, pea2 Δ, bud6 Δ, and bni1 Δ, also affect cell fusion and bipolar budding. However, another bipolar budding mutant, rvs161 Δ, exhibited no defects in Kar9p localization. In addition, Kar9p localization was not altered in the cell fusion mutants fus1 Δ and fus2 Δ. Thus, alterations in Kar9p localization are not due to general disruptions in either bipolar budding or the cell fusion process. Several other observations speak to the specificity of the effects of these mutations on Kar9p localization. Bnr1p is another formin family protein that shares overlapping functions with Bni1p . Kar9p localized normally in bnr1 Δ mutants (data not shown) and also did not show any genetic interactions with kar9 Δ or dhc1 Δ, indicating that the effects on Kar9p localization are not general to all formin proteins, but specific to Bni1p. Other mutants such as sla1 Δ that affect the assembly of the actin cytoskeleton also did not alter Kar9p localization in shmoos. This argues that the specialized interaction of Spa2p, Pea2p, Bud6p, and Bni1p with the actin cytoskeleton is the important feature for Kar9p localization. The localization patterns for Spa2p and Pea2p are interdependent, suggesting that their functions are closely interrelated . Consistent with this, we find that GFP-Kar9p mislocalization was very similar in the spa2 Δ and pea2 Δ mutants. Thus, with respect to Kar9p localization, these two mutants can be referred to as the “ spa2 class.” Interestingly, the spa2 class showed a dramatic difference in the severity of Kar9p mislocalization comparing shmoos and mitotically dividing cells. In mitotic cells, mislocalization was apparent in only ∼10% of cells. In contrast, in most shmoos, Kar9p localization was altered from a single dot to a cap-like pattern. Thus, Spa2p and Pea2p likely play very different roles in polarization and microtubule orientation in mating cells. The finding that Kar9p localization is often spread out over a broader area in spa2 Δ or pea2 Δ shmoos is consistent with the proposed role for Spa2p and Pea2p in restricting the zone of polarization in shmoos . One prediction from such a model would be that a narrowed site of polarization would restrict the area of microtubule interaction with the cortex by restricting Kar9p localization to a dot. Consistent with this, we found a “splayed-out” microtubule pattern in a subset of spa2 Δ mutants. Two models might explain the altered patterns of Kar9p localization. In one model, Spa2p and Pea2p may play a direct role in limiting Kar9p localization to a tight focus by helping aggregate the Bni1p/Kar9p complex. Alternatively, Spa2p and Pea2p might play an indirect role in Kar9p localization via their effects on shmoo morphology. In either case, correct polarization might be required to define a specialized region of the mating projection that is primed for cell and nuclear fusion. Such a primed region would facilitate the alignment of cytoplasmic microtubules, allowing nuclear congression to follow rapidly after cell fusion. In spa2 Δ and pea2 Δ mutant shmoos, the primed region would be more diffuse over the blunter mutant shmoo projection. In support of the indirect model, in spa2 Δ prezygotes the clustering of vesicles along the zone of cell fusion is disrupted . Regardless of the specific mechanism, these results highlight the differences in cell polarization between mating and mitotic cells. In contrast, both the bni1 Δ mutation and actin depolymerization resulted in strong defects in Kar9p localization in both shmoos and mitotic cells. Although the bni1 Δ mutant was defective for nuclear migration in both shmoos and mitotic cells, there were differences in the effects on microtubule orientation. In bni1 Δ shmoos, the microtubules were clearly misoriented, whereas mitotic cells did not exhibit an obvious defect. One interpretation might be that correct Kar9p localization is more important for microtubule orientation in mating cells than in mitotic cells. Alternatively, the difference between the two cells may arise from the different patterns of mislocalized Kar9p. In the bni1 Δ shmoos, GFP-Kar9p was present in a diffuse speckled pattern. In the bni1 Δ mutant, GFP-Kar9p mislocalized to secondary sites at the SPB and bud neck, where it might still be able to provide some orientation to the microtubules. Abundant evidence has shown that Bni1p, specifically, and formins, generally, are involved in organizing the actin cytoskeleton. In addition to Spa2p and Bud6p, Bni1p interacts with profilin and the small G-protein Rho1p . It seems likely that formins may play a general role in linking the actin and microtubule cytoskeletons. For example, mutations in the Drosophila formin, cappuccino result in mislocalized molecular determinants and abnormal microtubule patterns . In conclusion, we report here that Kar9p localization is dependent upon actin and the polarization protein, Bni1p. This provides the first evidence in yeast for a functional link between the actin and microtubule cytoskeletons. These data suggest a mechanism by which the polarized information contained within Kar9p is transferred into the vectorial movement of spindle orientation and nuclear movement into the bud. This work provides an avenue into future investigations as to how nuclear positioning is coordinated with the growth of the bud and cytokinesis. | Study | biomedical | en | 0.999996 |
10085295 | The haploid strains 9d ( MAT α , lys2–801 , his3–200 , ura3–52 , leu2–3, 112 ) and 8d ( MATa , lys2–801 , trp1 Δ, ura3–52 , leu2–3, 112 ) were parent strains for all experiments. 9d and 8d were transformed with the plasmids containing the fusion proteins listed below to produce cells with dynein–GFP, tubulin–GFP, or Nuf2–GFP. Mating studies were performed using transformed 9d cells mated to an a type mating tester ( MATa, ade6 ) that did not contain any GFP fusion proteins. In some experiments, alpha factor ( Sigma ) was used at 10 μg/ml and applied for 1–2 h to liquid 8d cultures in order to produce cells with visible shmoo growth. A galactose inducible dynein–GFP fusion protein was constructed by introducing GFP (S65T) into the COOH terminus of the cytoplasmic dynein heavy chain gene . Cells were grown to midlogarithmic growth phase in glucose, followed by a short induction on galactose (∼2 h). Aliquots of cells were removed and placed onto a 5-μm-thick, 25% gelatin slab containing 2% glucose . Expression was repressed for the duration of recording to restrict analysis to existent dynein. Also used in this study were a GFP–tubulin (gift of A. Straight, Harvard Medical School, Boston, MA) and Nuf2p–GFP . These two fusions were separately integrated into the genome of strain 9d or 8d and constitutively expressed by the native promoter for the given protein. Cells were selected for imaging as described by Shaw et al. by identification of faint green fluorescence at 75× magnification to the camera using 100% of the fluorescence excitation light at 490 nm. Variable levels of dynein–GFP were observed in all transformed strains. After increasing magnification to 150× and attenuating the fluorescence excitation to <10%, a single 3-s exposure was taken and compared with a fluorescent reference standard imaged under the same conditions. Cells containing an average fluorescence of less than 20% relative to the reference fluorescence (arbitrary units) were selected for time-lapse observation. Imaging was performed using the microscope system described by Salmon et al. and modified by Shaw et al. . Fluorescence emission from 3-s exposures was collected through a 530 ± 15 nm band-pass filter. A total of five fluorescence images were acquired at a z-distance of 1 μm between each image. A single differential interference contrast (DIC) image was made at the middle z-step by rotating the analyzer into the light path and taking a 0.6-s exposure. The entire acquisition regime was repeated once every 60 s for time-lapsed imaging. For measurements of Mt dynamics, three exposures of 1-s duration were taken at 100% fluorescence excitation at 1 μm axial steps. This acquisition regime was repeated every 15 s for a maximum of 15 min. A single background image constructed by averaging 24 3-s exposures with no illumination was subtracted from each GFP image to remove noise introduced by the camera. The five (or three) images corresponding to a single time-lapse point were projected to a single image by using only the brightest pixel at any one location in all five image planes. Registration of DIC and fluorescence images was verified by imaging of 1-μm fluorescent beads in DIC and fluorescence modes and determining displacement using the Metamorph software package. For presentation, dynein–GFP and corresponding DIC images were overlaid or placed side by side and contrast enhanced using Metamorph ( Universal Imaging ) or Photoshop (Adobe Systems). Images for publication were scaled and interpolated to 300 dots per inch. Average fluorescence and maximum fluorescence data were collected by creating a region of interest around a cell in the reconstructed image using quantitation tools in the Metamorph software package. Average and maximum fluorescence values were compared with data from a fluorescence reference slide (Applied Precision) imaged under identical conditions. Rates of Mt elongation and shortening were determined by measurement of Mt length in time-lapsed image sequences. Mt length was determined by measuring individual Mts from the center of the SPB fluorescence to the Mt end and converting from pixels to microns using an image of a stage micrometer. Where necessary, the original five (or three) planes were used to identify dynein–GFP-labeled Mts that moved through more than one focal plane. This technique is described in detail elsewhere , as are applications of fluorescence speckle microscopy (FSM) . In brief, FSM is a new method for visualizing the movement and sites of assembly of Mts in living cells. Fluorescent speckles in the Mt lattice originate by coassembly of a small fraction of fluorescently labeled subunits in a pool of unlabeled subunits. Random variation in subunit association creates a nonuniform fluorescent speckle pattern along the Mt. For our yeast application, we used a fusion to the amino terminus of the major alpha tubulin of yeast (Tub1p) with GFP. This labeled tubulin does not complement a tub1 deletion . However, when the native Tub1p and GFP–Tub1p (GFP– tubulin) are coexpressed, cells grew and divided with wild-type kinetics. The GFP–tubulin in these studies was expressed from a copy of the endogenous Tub1 promotor. The level of GFP–tubulin produced in the cells was sufficiently low in a percentage of cells to create fluorescent speckles in the Mt lattice. To induce the mating response, a GFP–tubulin-expressing strain of 9d was mixed with an a type mating tester strain. Cells were observed by bright-field and epifluorescence microscopy on a Nikon E-600 FN fixed stage microscope equipped with a 100×/1.4 NA Planapochromatic lens and a Chroma HiQ FITC filter set. Cells observed to be shmooing were imaged using both bright-field and fluorescence modes. Imaging and image processing was similar to that used for dynein–GFP cells with the following exceptions. Time-lapse intervals ranged from 1 to 15 s and single focal planes were acquired in lieu of z-sections. Exposure times ranged from 200 to 800 ms. The images were acquired by a Hamamatsu c4742-95 Orca camera controlled by Metamorph imaging software ( Universal Imaging ). Images were not binned in order to retain diffraction limited spatial resolution at 100× total magnification to the camera. For photobleaching experiments, cells containing constitutively expressed tubulin–GFP were combined with a mating tester strain (see above) of opposite mating type and prepared for imaging as described above for dynein–GFP. Techniques for marking fluorescent Mts by Argon laser photobleaching (488 nm wavelength) are well established . In this study, time-lapse epifluorescence images were obtained on a Zeiss AT model 100 inverted microscope ( Carl Zeiss ) using a 100×/1.3 NA Plan Fluor objective lens, a fluorescein filter set, and a 100-W Hg epifluorescence illuminator with no attenuation. Images were acquired through a Princeton Instruments MicroMax cooled charge-coupled device camera ( Princeton Instruments ) mounted on the bottom port of the microscope at the primary image plane. Custom time-lapse software called Phatlapse controlled the excitation shutter (Vincent Associates [VA]), filter wheel (Ludl Electronic Products [LEP]), z-motor stepping (LEP), image acquisition ( Princeton Instrument 's WinView software), laser shutter (VA) for photobleaching, and a motorized mirror (New Focus) for introducing the photobleaching laser light into the epiillumination light path. A stack of fluorescence optical sections was acquired at 15-s intervals in time-lapse mode. Each stack consisted of five 250-ms exposures taken 0.75 μm apart in the z dimension. Targeting of the laser on the specimen was done using an intensified silicon intensifier tube (ISIT) camera to view the fluorescent specimen at very low excitation light levels on a video monitor. A cylindrical lens was used with the objective to focus the laser into a bar ∼0.3 μm wide on the fluorescent bundle of Mts oriented toward the shmoo tip. Laser exposure times varied from 200 to 800 ms for photobleaching. We used GFP protein fusions and time-lapse digital imaging microscopy to investigate the mechanism of formation and maintenance of the Mt bundle to the shmoo tip. Haploid cells containing dynein–GFP, Nuf2p–GFP, or GFP– tubulin were mated to nonexpressing cells of opposite mating type. Fig. 2 shows two cells expressing dynein–GFP mating to nonexpressing partner cells. In late-telophase and G1 cells, astral Mts exhibited dynamic instability, transiently growing to the cell cortex in directions opposite to nuclear movement in the cell . In G1 mating cells, there was a conical array of 4 ± 0.8 astral Mts per SPB which grew and shortened with an average rate of ∼0.5 μm/min . The number, dynamic properties, and behavior of Mts were similar to those reported for astral Mts and nuclear movement in vegetative G1 haploid and diploid cells . Astral Mts became distinctly oriented toward one region of a mating cell, often before visible growth of a mating (shmoo) projection . Identification of this site as the preshmoo tip was determined by subsequent time-lapse images. Mt interactions with the preshmoo tip cortex were often the first morphological manifestation of mating. The cell shown in Fig. 3 was observed to finish mitosis and then mate with a neighboring cell. After spindle disassembly, the nucleus moved around the G1 cell in a rotatory fashion as described above . Astral Mts showed no preference for any given cell quadrant. When the free ends of astral Mts came in contact with the preshmoo tip site, a persistent attachment was formed . A bundle of fluorescent astral Mts formed between the SPB and the shmoo tip as new Mt growth became orientated toward the shmoo tip . In cells with shmoo projections , the fluorescence of the bundle of Mts from the SPB to the shmoo tip was 3.5 ± 1.2 ( n = 7 cells)-fold greater than the average fluorescence of a single astral Mt. This ratio indicates an average of three to four astral Mts in the shmoo tip bundle. Shmoo tip Mt bundles were never observed to detach from the shmoo tip in over 300 min of recording before cell fusion and karyogamy. This persistence is much longer than the 3.5-min average lifetime of astral Mts with free ends (Table I ). Persistent attachment did not affect Mt dynamic instability . Not all astral Mts became attached to the shmoo tip and those unattached Mts continued to exhibit dynamic instability, growing and shrinking at 0.56 ± 0.25 and 0.68 ± 0.26 μm/min, respectively . Stable interactions (localization >3 min) of astral Mts in regions of the cortex outside the shmoo tip or preshmoo tip were exceedingly rare, as reported by Shaw et al. for vegetative cells. We have also examined the behavior of astral Mts when mating pheromone, α factor, was added to the medium to induce shmoo formation. In contrast to mating cells, high concentrations of α factor in the medium induce a default mating response . In α factor–treated cells, astral Mts mainly oriented toward the preshmoo and shmoo tip, and exhibited assembly behavior as in mating cells. The total number of astral Mts was not noticeably different from mating cells, but a larger fraction of astral Mts, often two or three, were not attached to and/or grew away from the shmoo tip (20% more free Mts as determined by fluorescence intensity, n = 15 cells for each condition). Miller and Rose and Read et al. reported similar results in fixed preparations for α factor–treated cells. Thus, α factor–treated cells appear to have less robust Mt attachment complexes at their shmoo tips relative to untreated mating cells. Maintenance of the shmoo tip Mt bundle to the shmoo tip plays a critical role in mating efficiency . We discovered that the bundle of Mts is dynamic. The SPB and attached nucleus oscillated toward and away from the shmoo tip as the attached bundle of Mts shortened and grew . The velocities of SPB moving toward and away from the shmoo tip were 0.5 ± 0.27 μm/ min ( n = 24). These rates are similar to those measured for nonshmoo tip Mts in mating cells and astral Mts in vegetative cells (Table I ) . Switching of the shmoo tip Mt bundle from growth to shortening (catastrophe) was accompanied by SPB and nuclear movement toward the shmoo tip and occurred at similar frequencies to that of free end astral Mts (0.25/min, Table I ). Shmoo tip Mt bundle switching from shortening to growth (rescue) resulted in SPB and nuclear movement away from the shmoo tip and occurred at a similar frequency to catastrophe (Table I ). Bundles never shortened completely and disappeared. The shmoo tip Mt bundle grew and shortened without detaching from either the shmoo tip or SPB. We could not determine whether all Mts in the bundle were attached to the shmoo tip because of limitations in resolution. Regardless, Mt bundle attachment to the shmoo tip and/or the SPB must be a dynamic attachment. To determine the sites of assembly and/or disassembly of the shmoo tip Mt bundle, we used two methods to produce fluorescent fiduciary marks within shmoo tip Mts. These experiments required the use of GFP–tubulin, which incorporates into the Mt lattice, instead of dynein–GFP, which decorates the outside of Mts and presumably can exchange with cytosolic dynein–GFP . FSM and fluorescence photobleaching of Mts containing GFP–tubulin were used to produce fiduciary marks in the Mt bundle (see Materials and Methods). For FSM, haploid cells expressing low levels of GFP–tubulin were observed in the early stages of mating and shmoo formation. Clear speckles (fiduciary marks) of bright and dark fluorescence intensity along the shmoo tip Mt bundles were observed by time-lapse imaging . In nine out of nine examples, fluorescent speckles did not exhibit any apparent net movement relative to the pole over the course of time-lapse acquisitions . Speckles were seen to disappear and reappear at the shmoo tip end of the Mt bundle as the bundle shortened and elongated . Similar results were seen for Mts marked by laser photobleaching . Fiduciary mark movement observations were confirmed by measurements of the position of fluorescent marks (measured at the center of a speckle for FSM and the edge of laser photobleached marks closest to the SPB) relative to the center of the SPB image . Straight lines were fit through mark-to-SPB measurements over time by the least squares method. A best-fit line with a slope of zero would indicate no net change in distance between the mark and SPB over time, i.e., a rate of 0 μm/ min. The average slope of the lines through the data was near zero: an average of 0.034 ± 0.069 μm/min ( n = 9) for speckle data and −0.048 ± 0.036 μm/min ( n = 5) for photomarking data (Table II ). Fluctuations of individual measurements about the best-fit lines, calculated by root mean square, were 0.169 μm for FSM data and 0.073 μm for photobleaching data (Table II ). Although we cannot exclude the possibility of small units of assembly/disassembly at the SPB, these fluctuations most likely represent the limit of accuracy in measurement of the position of the fiduciary marks relative to the SPB. During time-lapse image acquisition, the SPB and shmoo tip Mts moved laterally and in and out of the plane of focus. In addition, observed fluctuations are less than the 0.25 μm limit of lateral resolution . Thus, slight shifts in focus could change the apparent position of the marks and the center of the SPB, producing the fluctuations observed. Taken together, these measurements indicate that there was little or no Mt assembly or disassembly at the SPB. Therefore, the great majority or all changes in Mt length occurred by assembly/disassembly at the shmoo tip. To analyze Mt dynamics during karyogamy, cells expressing dynein–GFP were mated to cells without dynein–GFP and observed by time-lapse imaging. Cell fusion was visualized in DIC by the loss of the cell wall septum (CWS) where the two shmoo tips had touched, and in fluorescence by movement of cytoplasmic fluorescence from a cell expressing dynein–GFP into a nonexpressing partner cell. As this movement occurred, dynein–GFP was seen to redistribute to the SPB and astral Mts of the nonexpressing partner cell. Thus, there is a freely diffusible pool of cytoplasmic dynein–GFP relative to dynein-binding sites at the SPB or along astral Mts . Upon cell fusion, oscillations of the SPBs and nuclei became attenuated and the nuclei slowly moved together concurrent with shortening of the internuclear Mt bundle . From 12 different experiments, the average rate of SPB congression was 0.2 ± 0.07 μm/min. The nuclei did not move equal distances to the midpoint position between the two nuclei at the time of cell fusion. Such bilateral movement would be expected from force generation via overlapping Mts. Instead, each SPB and nucleus moved to the site of cell fusion as the Mt bundle shortened and sometimes oscillated back and forth. As seen in Fig. 6 , when one SPB (SPB L in this case) was farther away from the site of cell fusion than the partner SPB (SPB U), the karyogamy event was not bilaterally symmetric. SPB U was close to the CWS and remained in place while SPB L moved ∼5 μm to the site of cell fusion. Free-end astral Mts facing the site of cell fusion often were seen to grow past the opposing nucleus to the distal reaches of the partner cell . Free ends of these extended Mts exhibited dynamic instability. Astral Mts in zygotes were on average longer than those seen in vegetative cultures (Table I ) . Carminati and Sterns proposed that catastrophe is induced when Mts contact the cell cortex, therefore the increase in length could be a result of greater distance between the SPB and the cortex in zygotes. Long Mts were also often seen to bend or buckle as they grew or traversed the cellular periphery. However, astral Mt growth did not result in a detectable change in position of the nuclei and/or the SPBs, and the spindle poles remained oriented toward each other. In contrast, astral Mts were rarely seen to extend from a SPB back into the periphery of its original cell, indicating each SPB orients new Mt growth in the direction faced by the majority of its Mts. At the end of karyogamy the zygotic diploid nucleus forms by nuclear and SPB fusion . In our DIC time-lapse images, the bud emerged at the position of the SPB. This is in contrast to the situation in vegetative mitosis, where astral Mt search and capture mechanism is used to position the SPB and nucleus toward the neck of the bud . In 10 out of 14 cells, the SPB remained localized to the site of cell fusion. In four out of 14 cells the SPB moved away from the site of cell fusion (data not shown). In all cases bud emergence coincided with the position of the SPB . This result is consistent with the findings of Byers , who centrifuged zygotes before bud emergence to dislodge cytoplasmic and nuclear structures. The bud emerged from the site proximal to the position of the displaced SPB and nucleus. The use of GFP probes (dynein–GFP, GFP–tubulin, or Nuf2–GFP) allowed a kinetic analysis of events in the first diploid mitosis. Two major differences were observed in the first division versus vegetative mitosis. First was the ability of each SPB to acquire cytoplasmic dynein immediately after SPB separation. In vegetative cells, there is a temporal delay in the acquisition of dynein–GFP (and presumably astral Mts) to one of the SPBs after separation. This delay may be coupled to the Mt-based search and capture process that ensures one and only one SPB orienting toward the bud . In the first zygotic division, there was no temporal lag of dynein–GFP decorating each SPB after their separation. When two spots were distinctly visible (0.5 μm of separation), the fluorescence intensity of dynein–GFP was equal at both poles . The fluorescence intensity of Nuf2p–GFP and GFP–tubulin at each SPB was also equal after their separation (data not shown). Since our cell fusion data between cells expressing dynein–GFP and nonexpressing cells showed that free pools of cytoplasmic dynein–GFP are available , there must be a mechanism that prevents one SPB from immediately acquiring cytoplasmic dynein in vegetative mitosis. The strategies for Mt nucleation from SPBs in zygotic division are therefore distinct from vegetative cycles. Secondly, once the SPBs began to separate, they became aligned along the bud tip–neck axis and, after a few minutes of delay (6 ± 2 min, n = 10) at a distance of 0.5–1 μm, there was a linear progression in spindle elongation (0.75 ± 0.3 μm/min, n = 10) to a 10–12-μm spindle characteristic of telophase. The velocity of spindle elongation was similar to that of the initial phase of anaphase spindle elongation in wild-type vegetative cells . In Fig. 8 , data for Nuf2p–GFP is shown; velocities from dynein–GFP and GFP–tubulin were similar. In contrast to this first zygotic mitosis, the preanaphase spindle (1–1.5 μm) persists for at least 20 min in the vegetative growth cycle. The graphs in Fig. 8 B begin when two spindle poles in each case were clearly resolvable and continue until the first rapid stage of anaphase was completed. In the first zygotic division after mating, the entire time from spindle pole separation to the initiation of anaphase spindle elongation was less than 6 min. This is much shorter than the 20-min period during mitosis in vegetative cells . Fig. 9 shows the kinetics of the major stages of the mating pathway . These results extend from the time of cell fusion to cytokinesis during the first cell division. Time of shmoo formation could not be accurately assessed due to the subtle nature of the early shmoo when viewed by DIC. The entire process took 179 ± 34 min ( n = 3). Our results show that localization of the nucleus to the preshmoo or shmoo tip occurs via Mt growth and shortening in a random search and capture process much as described for vegetative G1 cells . Once astral Mts encounter the shmoo tip, subsequent Mt growth results in formation of a shmoo tip Mt bundle that tethers the SPB and nucleus to the shmoo tip. The shmoo tip Mt bundle remains dynamic and represents a new model system to study mechanistic aspects of dynamic Mt attachments. The velocities of oscillatory movements of the SPB and nucleus toward and away from the shmoo tip equal the intrinsic growth and shortening velocities of free Mts. Thus, the shmoo tip is able to maintain an association with Mt ends without compromising Mt dynamics. The fluorescent intensity of the shmoo tip bundle allowed us to use photomarking experiments and fluorescence speckle microscopy to determine which end of the Mt was active in subunit addition and subtraction. The plus end (the end at the shmoo tip) was the main site of Mt bundle growth and shortening . Therefore, at least one Mt plus end was dynamically attached to the shmoo tip at any given time, otherwise there would have been detachment and loss of the bundle during oscillations. Dynamic attachments are a specialized means for harnessing Mt plus end growth and shortening to produce force needed for cellular processes. One well-studied correlative to the shmoo tip–Mt interaction is kinetochore dynamic attachment and movement along polar spindle Mts in vertebrate tissue cells. After attachment to plus ends of polar Mts, kinetochores oscillate toward and away from the pole, coupled to shortening and growth of their kinetochore fiber Mts mainly at the kinetochore . Kinetochores in budding yeast can also oscillate relative to their poles , but little is yet known about kinetochore Mt dynamic attachments. The dynamic Mt tip attachment complexes at the shmoo tip described herein may be functionally similar to the dynamic attachment of kinetochores to spindle Mts in tissue cells . Both kinetochores and shmoo tips exhibit multiple dynamic Mt attachments. In the case of the kinetochore, kinetochore Mts apparently polymerize/depolymerize in concert, resulting in oscillatory movement of the kinetochore and associated chromosome away or toward the spindle pole. How this is regulated is not yet known . A similar mechanism could be acting at the shmoo tip, where multiple Mts polymerize/ depolymerize coordinately, moving the SPB and attached nucleus away and towards the shmoo tip. Identification of shmoo tip molecules required for anchoring Mt plus ends may therefore provide fundamental insights on the mechanistic bases of dynamic attachments. What are the molecules in the shmoo tip Mt dynamic attachment complex? Candidates must (a) localize to the shmoo tip, (b) bind dynamically growing and shortening Mt plus ends, and (c) link structural factors (such as actin) at the shmoo tip to Mt plus ends. Examples of the many proteins that localize to the shmoo tip include Bni1p, a member of the formin protein family and a putative cortical protein, Kar9p . In particular, mutations in Kar9p and the Mt-binding protein Bim1p have been shown to disrupt Mt localization to the shmoo tip without visibly affecting shmoo formation . It is not known whether these proteins are involved directly in Mt attachment (Kar9p), Mt dynamics (Bim1p), or indirectly (Bni1p) by linking Mt attachment complexes to actin. In vertebrate kinetochores, Mt motor proteins are thought to couple kinetochores to dynamic Mt plus ends . Kar3p is a candidate for this job since it is a minus motor localized to astral Mts in the shmoo and the only known motor protein required for karyogamy . Time-lapse imaging of cell fusion and karyogamy revealed that nuclei do not simply move toward one another, but meet at the site of cell fusion. One explanation for this observation is that the septum aperture is too small to allow nuclei to pass through, therefore the two nuclei fuse at the site of cell fusion. However, the diameter of the mother–bud neck during anaphase is also ∼1 μm and does not seem to inhibit nuclear movement. Alternatively, the site of cell fusion may provide a landmark for subsequent events. Mt plus ends that were contained within the bundle of Mts between nuclei shortened as the nuclei moved to the site of cell fusion, whereas those not in the bundle grew past the nucleus to the end of the opposite cell where their plus ends exhibited dynamic instability. Perhaps the shmoo tip Mt attachment molecules persist in their attachment to Mt plus ends after cell fusion and these complexes are involved in maintaining bundle formation and producing or regulating Mt disassembly and nuclear movement. Our kinetic analysis shows that the nuclei move together as the bundle of Mts shortens at rates much slower (0.2 μm/min) than the in vitro translocation velocity of Kar3p . This slow rate does not exclude a role for Kar3p in pulling together interdigitating Mts from each haploid nucleus. However, karyogamy is clearly not limited by the rate of motor activity, but appears more tightly coupled to mechanisms that regulate Mt bundle disassembly. Meluh and Rose proposed that at cell fusion, plus end Mt dynamics are stabilized whereas Kar3p, acting at the SPBs, induces minus end disassembly therefore drawing the SPBs to the site of cell fusion. Our kinetic data is consistent with but does not prove this idea. After fusion, oscillations in the length of the Mt bundles ceased as if Mt plus end dynamics were stabilized . Bundle shortening occurred at ∼0.2 μm/min. This rate is consistent with the predicted rate of Kar3p depolymerization of minus ends located at the SPBs based on the observed rate of Kar3p depolymerization of taxol stabilized Mt minus ends in vitro . Kar3p depolymerizing Mt minus ends at a rate of 0.1 μm/min at both poles would indeed yield a karyogamy rate of 0.2 μm/ min. However, we did not detect any astral Mt minus end assembly/disassembly at the SPB before cell fusion. We have compared the kinetics of mitotic SPB separation and spindle elongation in zygotes versus vegetative cells . The kinetics of Mt nucleation from separated SPBs, as well as the rate of spindle elongation in the first zygotic division reported herein are remarkably distinct from vegetative mitosis. As observed by Shaw et al. , dynein–GFP is delayed in decorating the SPB destined to the mother cell in vegetative mitotic cells. Since we now know that there is a freely diffusible pool of dynein–GFP , it is now more certain that the absence of dynein–GFP at the spindle pole reflects a temporal delay in acquisition. This delay was hypothesized to promote spindle orientation along the mother–bud axis, and unity in pole deposition into the bud . In the first zygotic division , two poles are detectable by dynein–GFP with the same kinetics as Nuf2p–GFP . Since bud emergence coincides with the position of the SPB, the need for search and capture is obviated. These differences in Mt nucleation and nuclear movement are the first indications that regulatory aspects of the zygotic division are not equivalent to those found in vegetative growth. Analysis of spindle elongation kinetics illustrates the third substantial difference in the regulation of zygotic mitosis relative to vegetative cells. As soon as the SPBs can be resolved, they become oriented along the bud tip–neck axis. As the poles align along this axis, spindle elongation ensues, unlike the vegetative cycle where the 2-μm spindle persists for ∼20 min ; the 2-μm stage of spindle morphogenesis in the zygote is very short lived. Once spindle elongation begins, it occurs with linear kinetics (0.75 μm/min) until the SPBs reach the distal ends of the mother and first bud. The kinetics of spindle elongation and seeming lack of time spent at the 2-μm spindle stage may reflect, in part, the fate of proteins at the shmoo tip. One intriguing possibility is that proteins at the two shmoo tips form a midshmoo body, analogous to the midbody in tissue cells. In this scenario, proteins from the shmoo tip, which are also markers of bud site selection, accumulate at the interdigitating Mts (midbody). This hypothesis provides a mechanism by which the position of the SPB directs the position of the first bud. Such a mechanism may obviate the requirement for a search and capture mechanism using Mt growth dynamics to find the bud. In evolutionary terms, bypassing the search and capture process expedites the cell's ability to form a diploid population, and therefore alleviate the need for certain cell cycle checkpoints. The above results illustrate that the first zygotic division in yeast is distinct from vegetative mitosis. Kinetic analysis of mating and the ability to monitor Mt dynamics throughout the entire mating process reveals a new model system for studying Mt plus end interactions with cellular structures, and cell division in budding yeast that is analogous to early embryonic divisions of larger eukaryotes, such as the frog Xenopus laevis . The question that remains is why early embryonic divisions, now from the first zygotic division in yeast to the thousands of divisions in a mammalian blastula, dispense with mitotic controls that are prominent in vegetative cells and the cells of our body. | Study | biomedical | en | 0.999999 |
10085296 | Isolation of dominant suppressors of heldup 2 was described in Prado et al. . In brief, adult hdp 2 males were mutagenized with ethyl-methane sulfonate (EMS) according to standard procedures, and crossed to females of the genotype C(1)M3/Y ; Sco/CyO or C(1)M3/Y ; TM1/TM3 . Male offspring with near normal wing position, instead of the expected heldup wings, were crossed individually to balancer stocks to identify the chromosome containing the suppressor. Stocks with a series of recessive markers were used to determine the map position of each suppressor on a particular chromosome based upon recombination between markers. Each isolated suppressor should be designated as Su(hdp 2 )D followed by an identification number. For brevity, they are referred to as D mutations in the text. As per standard practice, gene abbreviations are designated in italics and proteins are in capital Roman type. We obtained recessive–lethal, homozygous suppressor strain embryos for DNA amplification and sequencing by using a second chromosome balancer line (CyO y + ) marked with the yellow + gene in combination with an X chromosome marked with the y and w (white eye) mutations. To this end, hdp 2 ; D mutation/ CyO males were mated with y w ; CyO y + /Bc Elp females. Male offspring of genotype y w ; D mutation/ CyO y + were backcrossed to y w ; CyO y + /Bc Elp females. Resulting males and females of the y w ; D mutation/ CyO y + genotype were mated to produce a stable stock. Embryos with dark mouth hooks carry one or two copies of the second chromosome marked with CyO y + , while homozygotes for the D suppressor mutation display yellow mouth hooks. Genomic DNA was extracted from homozygous embryos of each suppressor mutant according to the method of Jowett . 60 embryos were frozen in an Eppendorf tube and stored at −80°C for at least 1 h. 40 μl of single fly homogenization buffer (10 mM Tris-HCl, pH 7.5, 60 mM NaCl, 50 mM EDTA, 150 μM spermine, 150 μM spermidine) were added and the samples were ground with a plastic pestle. 40 μl of single fly lysis buffer (1.25% [wt/vol] SDS, 300 mM Tris-HCl, pH 8, 100 mM EDTA, 5% [wt/vol] sucrose, 0.75% freshly added diethyl pyrocarbonate) were added. The mixture was incubated for 30 min at 60°C. The sample was cooled to room temperature and 12 μl of 8 M potassium acetate was added. After cooling on ice for 45 min, debris was pelleted by 1 min centrifugation in a microfuge. Supernatant was removed to a fresh tube and 200 μl of 100% ethanol was added. DNA was precipitated at room temperature for 10 min and pelleted in a microfuge for 10 min. The sample was washed with 80% ethanol and vacuum dried. The pellet was resuspended in 60 μl TE (10 mM Tris-HCl, pH 8, 1 mM EDTA). Genomic DNA from each mutant was used in PCR to generate 11 fragments that cover the entire coding region, plus flanking introns of the Mhc gene. The following oligonucleotide primers were used for amplification (sequences given for noncoding strand in a 5′ to 3′ orientation): 1, ATGCCGAAGCCAGTCGCAAAT , GGAATTCGATACGGATGAATTTACC ; 2, TAAGCTTGAAGACCGATGAGGCC , ATAGCCGTCACTACATAGAGC ; 3, TTATGTTCTTCTTGCTAAACC , ATCTGACTAAAATCCTCAGA ; 4, GATACACTGCAGCACTAT , TGATCGGAGGCCTTGGGGAAC ; 5, GTTCCCCAAGGCCTCCGATCA , GTGTGGGGATTCAATTGAAAG ; 6, GGAATCAAAAACGAACTCTAC , CTAATTGTGGAAGGAGC ; 7, GTTAAGATCAACTGTAACTAA , AGACCCAGGCTGGTCTCGTT ; 8, CTTCAGCCCGAATCGACCGCC , TCAGATCTCTCTATCTCGAT ; 9, TTGAAGGATCTACAGTTTACA , GGGTGACAGACGCTGCTTGGT ; 10, GTCCCAGGTGTCTCAGCTGT , GGCGGGCGGCATCGACCATAG ; and 11, TGCGTCGTGAGAACAAGAACC , TATTACTCTCTTGTTTT . Each PCR sample contained 5 μl of genomic DNA, 20 μl of 10× PCR buffer ( Promega Corp. ), 20 μl of 5 μM solutions of each dNTP (80 μl total), 16 μl of 25 mM MgCl 2 , 100 pmol each of two primers, 0.8 μl of Taq polymerase ( Promega Corp. ), and was brought to a total volume of 200 μl with distilled H 2 O. Paraffin oil was placed on top of the sample to prevent evaporation, and DNA was amplified in an Ericomp thermocycler as follows: one cycle at 95°C for 1 min, 45°C for 2 min, 72°C for 40 min; 28 cycles at 95°C for 1 min, 45°C for 2 min, 72°C for 6 min; and one cycle at 95°C for 1 min, 45°C for 2 min, 72°C for 15 min. Paraffin oil was then removed and DNA was chloroform extracted and precipitated. PCR products were cloned before sequencing. Amplified products were separated by agarose gel electrophoresis, isolated using GeneClean (Bio 101), and blunt ends were created with the Klenow fragment of Escherichia coli DNA polymerase I . Each fragment was cloned into the EcoRV site of pKS plasmid (Stratagene) and DNA sequencing was performed using a Sequenase kit (United States Biochemicals) or on an automated DNA sequencer (Applied Biosystems). First strand synthesis of cDNA was performed using 1 μg of total RNA , 100 pmol of 3′ primer , 1.4 μl of 5× first strand buffer (250 mM Tris, pH 8.5, 375 mM KCl, 5 mM MgCl 2 , 50 mM dithiothreitol), brought to a total volume of 7 μl with distilled H 2 O. The mixture was placed in boiling water for 30 s, then allowed to cool to 37°C. 1 μl of Inhibitase (1 U/μl; Promega Corp. ) was then added along with 0.5 μl of each dNTP at 10 mM. Then 0.6 μl of 5× first strand buffer was added plus 0.5 μl of distilled H 2 O. The reaction was started by addition of 1.0 μl of M-MLv reverse transcriptase (100 U/μl; GIBCO BRL ) and the sample was incubated at 37°C for 1.5 h. The reaction was terminated on ice by adding 20 μl of 0.3 M NaOH/0.03 M EDTA. RNA was hydrolyzed at 60°C for 1 h. The solution was neutralized by adding 3.4 μl of 3 M sodium acetate, pH 5.2, and cDNA was precipitated with 2.5 vol of 100% ethanol. After centrifugation in the microfuge for 15 min at 4°C, the DNA pellet was washed with 80% ethanol and vacuum dried. The sample was resuspended in 10 μl distilled H 2 O. Half the sample was amplified using the 3′ primer at position 10131 and 5′ primer GGCTGGTGCTGATATTGAGA , as described for genomic DNA above. Slides were cleaned by thorough washing with liquid hand soap, then treated with subbing solution (0.5% gelatin, 0.05% chrome alum). Slides were dried overnight in a dust-free environment. Tissue was prepared by embedding whole flies (with wings removed) in OCT compound and freezing on dry ice. Frozen tissue sections (8–16 μm) were taken using a microtome. These were placed onto treated slides and allowed to dry. Tissue was fixed with 4% paraformaldehyde for 20 min and then washed three times in 1× PBT (1.3 M NaCl, 0.07 M Na 2 HPO 4 , 0.03 M NaH 2 PO 4 , 1% Tween 20). Sections were then treated with 50 μg/ml proteinase K in PBT for 3 min. This was followed by treatment with 2 mg/ml glycine in PBT for 1 min (repeated once). Slides were washed in PBS (1.3 M NaCl, 0.07 M Na 2 HPO 4 , 0.03 M NaH 2 PO 4 ) for 1 min and placed in 4% paraformaldehyde for 20 min. This was followed by two washes with PBS for 5 min each. The samples were dehydrated in 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol (5 min each), and placed under the vacuum for 40 min. Transcription of digoxigenin-labeled probes was according to the procedure provided in Genius 3 Kit ( Boehringer Mannheim ). Antisense probes from each copy of exon 7 were prepared from the following fragments that had been cloned into a plasmid containing a T3 or T7 RNA polymerase binding site: exon 7a, XbaI to HindIII ; exon 7b, HindIII to HindIII ; exon 7c, Hind III to EcoRV ; exon 7d, EcoRV to EcoRI . 1 μg of RNA probe was added to 25 μl of 10 mg/ml tRNA and brought to a total volume of 100 μl with distilled H 2 O. The probe was denatured by heating at 75°C for 10 min. Hybridization was carried out by adding the denatured probe to 400 μl of hybridization buffer (50% formamide, 10% dextran sulfate, 0.3 M NaCl, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.1% Tween 20, 50 μg/ml heparin, 1× Denhardt's solution). 100 μl of the probe in hybridization solution were placed onto each slide. Slides were covered with a plastic sealer (HybriWell, Research Products International) and placed in a sealed box. Hybridization was allowed to proceed for at least 18 h at 56°C. After hybridization, slides were washed with 4× SSC (twice for 10 min each). This was followed by RNase A treatment (20 μg/ml in 0.5 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA) to remove single-stranded probe for 30 min at 37°C. Slides were washed in PBT for 5 min (repeated once), and then incubated with antibody conjugate at a ratio of 1:500 in PBT plus 5% normal goat serum for 120 min. Unbound antibody was washed off with buffer 3 (100 mM Tris, pH 9.53, 100 mM NaCl, 50 mM MgCl 2 ) for 5 min. This was repeated. Color reaction buffer was prepared by adding 20 ml of buffer 3 to 100 μl of NBT and 75 μl of X phosphate. This reaction was allowed to proceed for at least 1 h and as long as overnight. The reaction was stopped by rinsing in H 2 O. One-dimensional SDS-PAGE was performed by the method of Laemmli . Upper thoraces from 10 flies were dissected, homogenized in 100 μl sample buffer and boiled. Samples (10 μl) were loaded on gels containing 9.5% acrylamide. After staining in Coomassie blue, scanning was performed using a Molecular Dynamics densitometer. MHC levels were normalized to actin levels within the same lane to account for differences in protein loading levels. Flight testing was performed using the method of Drummond et al. on young (2-d-old flies). For transmission electron microscopy, flies were dissected according to the protocol of Peckham et al. . Once the heads, wings, and abdomens were removed, thoraces were fixed overnight at 4°C in 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. The dorsolongitudinal muscles (DLMs) were dissected from the thoraces, washed several times in buffer, and postfixed in 2% OsO 4 in buffer for 45 min at 4°C in the dark. After dehydration, DLMs were embedded in Araldite resin. Silver sections (60–70 nm) were cut on a Reichert Ultracut E ultramicrotome, collected on Formvar-coated grids, and counterstained with uranyl acetate (10 min) and lead citrate (10 min). Micrographs were obtained using a JEOL 1200 EX electron microscope. Morphological analysis at the light microscope level was carried out on paraffin-embedded samples stained with Toluidine blue . The DLMs are composed of six fibers (a–f) attached to the anterior and posterior sides of the thorax . The DLM fibers, like the opposing dorsoventral indirect flight muscles, are termed fibrillar muscles. This is because each fiber contains several hundred myofibrils that can be easily teased apart. Individual fibrils are subdivided by transverse bands of electron dense material, the Z bands, that define the unit of contraction, the sarcomere . In a transverse view, the circular fibril contains a crystalline-like array of thick and thin filaments that is arranged in a 1:6 hexagonal pattern . In the normal strain used here, Canton-S (CS), ∼1,000 thick and 2,000 thin filaments accumulate in each fibril. These numbers are fairly constant within a muscle showing only a 5% variability in DLM muscle (a) of our CS stock. Note, however, that other normal strains may exhibit up to 1,500 thick filaments per fibril. In the troponin I mutant heldup 2 , the six DLMs appear torn apart from the center . In the remaining muscle material, near the attachment sites, the sarcomere length is 40% reduced and the thick–thin filament pattern is destroyed mostly due to the collapse of thin filaments . It appears as if the mutant muscles were clamped in a state of hypercontraction. The mutation hdp 2 is a single amino acid change, Ala 55 Val , affecting all known isoforms of troponin I . This corresponds to residue 25 in rabbit skeletal muscle troponin I . To identify molecular interactions between muscle proteins and troponin I, we screened for mutations that suppress the heldup wing position of the troponin I hdp 2 mutation and isolated four D mutations that map to chromosome II . We employed meiotic recombination to discern their locations on the second chromosome, and found they map between markers rd and pr . Further, we localized recessive lethality associated with mutations D41 , 45, and 62 to the interval uncovered by Df(2)H20 . This deficiency removes polytene chromosome regions 36A8–36A9;36F1 and contains the myosin heavy chain (Mhc) gene. To determine whether the suppressor mutations are Mhc alleles, we performed genetic complementation tests with known Mhc alleles . We crossed each of the D -suppressor mutants to a null mutant ( Mhc 1 ), a hypomorphic mutant ( Mhc 2 ), and several point mutants ( Mhc 5 , Mhc 6 , Mhc 8 ). Mhc 1 , Mhc 2 , and Mhc 8 are recessive lethal alleles, while Mhc 5 and Mhc 6 are viable as homozygotes. Our results show that the D -suppressor mutants are likely to be Mhc alleles, since none of the suppressors produced progeny over Mhc null or hypomorphic alleles, except for D1 which occasionally was viable in combination with Mhc 1 . The suppressors produced viable progeny in combination with the various point mutations, except that D1 is lethal in combination with Mhc 5 , D41 is lethal with Mhc 8 , and D62 produces very few viable adults in combination with Mhc 8 . These data demonstrate interaction, and likely allelism, between the D -suppressor mutants and Mhc . Since Mhc null alleles are recessive lethal , as are three of the four D -series suppressor mutants, it is important to determine whether the latter exert their suppression effect through failure to accumulate MHC. We determined whether MHC protein accumulates in the suppressor strains by crossing each to Mhc 10 and measuring MHC levels in upper thoraces of heterozygotes. Mhc 10 adults fail to accumulate MHC in the jump and indirect flight muscles due to a mutation in an alternative exon specifically used in these muscle types . Each of the D/Mhc 10 heterozygotes accumulate more MHC than Mhc 10 /Mhc 10 adults, but less than +/ Mhc 10 individuals (Table I ). This indicates that suppressor mutations produce stable MHC protein. While the suppressor mutants accumulate only ∼65–85% as much MHC as flies carrying one copy of wild-type Mhc gene, it is clear that suppressor alleles are not null mutations for Mhc . It is also noteworthy that Mhc missense mutations that cause flight muscle dysfunction typically result in less than wild-type levels of MHC accumulation . To demonstrate that each suppressor mutation resides within Mhc , and to determine their molecular lesions, we cloned and sequenced the Mhc gene from homozygous embryos of each strain. We found that each suppressor strain has a discrete region of the Mhc coding sequence altered, and all mutations affect the head domain (S1 fragment) of the myosin molecule. We mapped encoded aberrations onto the three-dimensional map of chicken myosin head . Amino acid identity between Drosophila and chicken myosin is high, and the atomic resolution crystal structure of the chicken molecule serves as an excellent model for visualizing Drosophila alternative coding regions and mutations . D1 is a point mutation in exon 10 (A→ G), changing amino acid 625 (chicken MHC numbering system) from Asp to Gly (Table I ). This mutation affects an amino acid at the base of the second loop of the molecule . This loop is involved in actin binding . If the mutation affects the mobility of the loop, it could dampen acto-myosin interaction. Mutation D62 also affects exon 10, and is a 24-bp in-frame deletion starting at amino acid 638 (Table I ). Like D1 , this mutation affects the loop that binds actin. It removes eight amino acids within the loop and clearly would be expected to affect actomyosin interaction. The loop, which runs from residue 627 to 646, is not visible in Fig. 2 due to its flexible nature . Mutation D45 is a point mutation in exon 5 (G→ A), changing amino acid 261 from Ala to Thr (Table I ). This amino acid is in the general vicinity of ATP entry and the ATP binding site . However, it is on the surface of the molecule, away from direct interactions with the nucleotide. It is located very close to loop 1 of the molecule (residues 204–216), which is not visible in the structure. This loop is important for regulating nucleotide entry and exit from the ATP binding pocket . Mutation D41 is a 2-bp insertion into exon 7a, interrupting amino acid codon 328. It places this alternative exon out of frame and inserts a stop codon (Table I ). The mutation also produces a potential 5′ splice junction, GTAGCT. This could disrupt alternative splicing. To study this, we used RT-PCR to amplify the exon 7 region in adult upper thoraces from this mutant. Since this mutation is recessive lethal, the thoraces were taken from D41/Mhc 10 organisms . We cloned the PCR products from D41/ Mhc 10 heterozygotes and analyzed a number of clones by DNA sequencing or restriction enzyme digestion. Normally exon 7d is used in indirect flight muscles , which make up the bulk of the thorax. We found this to be the case in all 17 clones analyzed from wild-type thoraces. However, we observed an extreme reduction in exon 7d usage, replaced by in-frame inclusion of exons 7b or 7c, in clones of Mhc PCR products from thoraces of D41/Mhc 10 organisms (1 exon 7b, 13 exon 7c, and 4 exon 7d). Thus, the insertion of a splice junction in exon 7a appears to disrupt the alternative splicing process. We next used in situ hybridization to investigate the possibility of tissue-specific alternative splicing disruption in thoracic musculature of D41 adults. Alternative exon-specific probes were prepared and hybridized to sections of young adults, either wild-type or D41/Mhc 10 mutant. The hybridization results clearly showed that exon 7d accumulates in indirect flight muscles of wild-type, but is below detectable levels in D41 indirect flight muscles . High levels of exon 7c accumulate in D41 indirect flight muscles, but no trace of this exon is detected in wild-type indirect flight muscle transcripts. Thus, the unusual effect of the mutation is to disrupt the alternative splicing apparatus through the introduction of a 5′ splice site, resulting in use of a different alternative exon than is normally employed in indirect flight muscles. Exon 7 encodes a region at the lip of the nucleotide binding pocket . It is possible that using the wrong version of this alternative exon disrupts MHC function by changing nucleotide affinity and disrupting the ATPase cycle. We also studied use of the aberrant version of exon 7a in the D41 mutant. In wild-type embryos, alternative exon 7a is abundantly expressed in body wall muscles (Zhang and Bernstein, manuscript in preparation). Our RT-PCR analysis of RNA from wild-type embryos confirmed that this is the major exon 7 version used at this stage (16 clones examined) and showed that exon 7a is incorporated in all reverse transcribed mRNAs studied from homozygous D41 embryos (14 clones). The normal splice junction is used in the mutant. This would result in premature termination of translation due to the stop codon described above, and explains the recessive lethality of the mutation. The suppressive effect of the D41 mutation upon the hdp 2 phenotype, however, appears to result from misexpression of exon 7c in the indirect flight muscles. We examined the degree of rescue of hdp 2 phenotypes by each suppressor mutation that maps within the head domain of MHC. While the suppressed wing position phenotype is evident in all hdp 2 ; D/+ males, none can jump or fly under standard criteria . We analyzed the structural effects of the suppressors in hdp 2 ; D/+ males at light and electron microscopic levels . In general, the organization of the six DLMs is restored with similar efficiency by the four D mutations. However, the e and f muscles, their posterior region in particular, are still very sensitive to contraction, and appear grossly abnormal at 3–5 d . Wild-type sarcomere structure and length in hdp 2 individuals is recovered to different degrees as a result of each D mutation. The M line reappears in all four cases but the sarcomere length is best restored by D41 . Organization of Z bands is better in D1 and D62 than with the other two alleles . The number of thick filaments per fibril averages 950 in D45 , 830 in D41 , 750 in D62, and 650 in D1 . These are 5–35% below normal. In spite of nearly normal numbers of thick filaments, D41 fibrils appear particularly unstable at the periphery, where the lattice collapses . These features, and those reported for second site suppressor D3 , point toward differential sensitivity of the center versus the periphery of the fibril. The arrangement of thick and thin filaments found in the suppressed condition include various types of abnormalities, e.g., absence of a thick filament, excess thin filaments, substitution of thick by thin filaments, or doublets of thick filaments . These perturbations do not induce major defects in the surrounding structure. We also studied the effects of the D -series suppressors upon flight muscle function in the absence of hdp 2 mutation. The suppressors show dominant effects upon flight muscle function (Table II ). D1 is least disruptive, with 85% of adults flying upward or horizontally, compared with 90% in wild-type. D62 is most disruptive, with only 16% flying upward or horizontally (Table II ). We determined whether the wild-type Mhc gene could rescue defects in flight ability by crossing each suppressor strain to a stock containing an Mhc transgene . No rescue was observed (Table II ), consistent with our observation that suppressor alleles produce stable MHC proteins which interfere with myofibril function. To investigate the unique nature of each suppressor's action, we tested all pairwise combinations of D mutants in a hdp 2 male background. We expect an additive or synergistic effect when two Mhc mutations are suppressed by different mechanisms. If the same mechanism of suppression is employed by two different suppressors, we expect a phenotype similar to that of flies with a single suppressor. Only combinations over D1 resulted in viable adults, and the structure of the resulting a or b fiber from their DLMs is illustrated in Fig. 5 . In the three cases of transheterozygotes, muscle structure is closer to normal than in each of the four independent D mutants. In addition, hdp 2 ; D1/ D41 flies are able to jump while the D/+ mutants are not. Interestingly, the D1/D62 combination exhibits a high number of double thick filaments. This abnormal feature is rarely seen in D1/+ or D62/+ muscles. The synergistic effects of D1 suppression observed in combination with each of the other alleles suggests that D1 employs a unique suppression mechanism compared with the other D -series Mhc alleles. Next, we tested whether other Mhc alleles are capable of suppression of hdp 2 phenotypes, either alone or in combination with D -series suppressors (Table III ). Three point mutations and the H20 deficiency chromosome were chosen to observe effects of specific amino acid changes or reduction in MHC levels upon the hdp 2 phenotypes. Homyk and Emerson had previously described a negative interaction between two of these alleles ( Mhc 5 and Mhc 8 ) and hdp 2 . Our data corroborated that Mhc 5 is lethal in combination with hdp 2 /Y , but showed a reduced viability, rather than complete lethality, between Mhc 8 and hdp 2 /Y (Table III ). The heldup phenotype was maintained in viable organisms in the latter case. This was also seen for the Mhc 6 point mutation and the deficiency chromosome. These results indicate that underexpression of MHC or non- D point mutations known to cause a dominant flightless phenotype do not suppress the heldup wing phenotype associated with specific troponin I allele hdp 2 . We studied the non-suppressor Mhc point mutants in more detail in an attempt to clarify their ability or inability to interact with the hdp 2 mutation. Each mutant accumulates substantial levels of MHC in adult thoraces: Mhc 5 homozygotes at 88% of wild-type levels, Mhc 6 homozygotes at nearly 100% , and Mhc 8 /+ (which is recessive lethal) at 79% . Mhc 6 is a point mutation (Arg to His) in the rod of the myosin molecule . We determined molecular defects in the other two mutants by sequencing clones containing PCR-amplified copies of their Mhc genes. As suspected, these mutations result from single amino acid changes. In the case of Mhc 5 , amino acid 200 is mutated from a Gly to Asp (resulting from an A to G transition in exon 4). On the three-dimensional crystal structure, this residue is located near the base of loop 1 of the molecule, at the beginning of a long helix that appears to interact with the bound nucleotide . Interestingly, the mutated amino acid in Mhc 5 is quite close to residue 261, which is mutated in suppressor strain D45 . The Mhc 8 mutation is located in the region that binds regulatory light chain, at residue 832 . The C to T mutation in exon 12 results in a change from Tyr to His. This portion of the molecule is part of the lever arm that is proposed to move during the myosin power stroke, due to pivoting about a point near the active site . These three Mhc point mutations exhibit very different effects when tested in combination with the D mutations in a hdp 2 background (Table III ). D1 is lethal when over Mhc 5 , but viable over the other two Mhc alleles and the deficiency chromosome ( Df(2)H20 ). In contrast, Mhc 8 is lethal or poorly viable over D41 , D45, or D62, but not over D1 . The Mhc 6 mutation has no effect on viability in combination with suppressor mutations or on their ability to suppress heldup wing phenotype, except for a reduction in suppression with the D45 allele. Finally, we tested the troponin I allele specificity of heldup wing suppression by D -series mutations. We used hdp 3 or hdp 2 / hdp 3 as alternative backgrounds. The hdp 3 point mutation causes abnormal RNA splicing, resulting in failure of a specific subset of troponin I isoforms to accumulate in the indirect flight muscles . hdp 3 mutants display a paucity of thin filaments and severely disrupted myofibrils . We detected no suppression in hdp 3 or hdp 2 / hdp 3 backgrounds, indicating that D -series alleles suppress a specific molecular defect in hdp 2 mutation. Taken together, our genetic studies demonstrate that suppression of the heldup wing phenotype in the hdp 2 point mutant can only result from specific modifications of MHC structure, as opposed to other perturbations in MHC structure or reductions in myosin concentration. Conversely, structural defects in DLMs caused by depletion of certain troponin I isoforms cannot be suppressed by these single amino acid changes in MHC. In this paper, we identified an unexpected interrelationship between myosin and troponin I through the use of a mutational screen for increased muscle function and integrity. We demonstrated that specific mutations in Mhc revert the heldup wings phenotype and muscle degeneration displayed by flies carrying the hdp 2 allele of troponin I. This reversion is allele specific, both for troponin I mutations and mutations in myosin, indicating that our approach identifies a novel type of functional interaction between the muscle proteins. Our data demonstrate that suppressive effects of D -series mutations do not arise simply from a reduction in myosin. This is based on accumulation of MHC in the mutant lines, as well as the failure of Mhc null mutations to suppress hdp 2 . The role of the amino acid mutated in hdp 2 may be inferred from recent structural and functional studies on this region of the protein in vertebrate troponin I. The hdp 2 mutation affects the NH 2 -terminal α-helical portion of the protein shown to interact with troponin C . Rabbit skeletal muscle troponin I/troponin C cocrystal structure shows hydrophobic interactions between residue 25, which corresponds to the site of hdp 2 mutation, and troponin C . Although interaction between troponin I and troponin C appeared stable , the NH 2 -terminal fragment is now proposed to be released upon Ca 2+ binding to troponin C . This release permits binding of an inhibitory domain of troponin I to troponin C, allowing the tropomyosin strand to move from its position blocking actin–myosin interaction. A reasonable model for hdp 2 defect is that the mutation hastens release of the α helix at lower Ca 2+ concentrations, resulting in more ready binding of troponin I's inhibitory domain to troponin C. Unregulated actin–myosin interaction would result. The hypercontracted sarcomeres and muscle degeneration observed are consistent with this model , as is the requirement for thick filaments for the degenerative phenotype . The four suppressor alleles within the Mhc gene may identify specific molecular interactions between troponin I and myosin. Direct interaction between the troponin complex and the myosin head in insect flight muscle is structurally feasible, since antibody labeling of troponin complexes show they occur at some sites of rigor crossbridge attachment . Myosin interaction may occur directly with the wild-type troponin I residue identified by the hdp 2 mutation, perhaps aiding release of the surrounding α-helical region during Ca 2+ binding by troponin C. This would facilitate actomyosin interactions, allowing the thin filament to progress to a fully active state. When poor regulation occurs in the hdp 2 mutant, the suppressor mutation could prevent or alter myosin interaction with the troponin I molecule. This would decrease the mutant troponin I's ability to release from troponin C, allowing the blocking action of troponin I on actomyosin interaction to continue at low Ca 2+ concentrations. More normal muscle structure and function would result. Thus, while the troponin I mutation could alter the equilibrium among the three states of the thin filament proposed by McKillop and Geeves and Vibert et al. , this equilibrium could be reestablished through a compensating mutation in the myosin head. The observation by Lin et al. , that troponin mutations can alter cycling of crossbridges, supports this possibility. Direct interaction between mutated residues in troponin I and the myosin head is feasible for the residues identified by the D62 Mhc mutation. Biochemical , structural , and chimeric molecule studies indicate that residues deleted from the actin binding loop of MHC in mutation D62 normally interact with the thin filament during the crossbridge cycle. For suppressor mutation D1 , changes in orientation of the actin-binding loop could result from amino acid alteration at the loop's base. Instead of revealing a direct interaction between troponin I and MHC, D1 or D62 could affect crossbridge cycling and indirectly compensate for the troponin I mutation. The mechanism of action of these two suppressors may be similar. However, the synergistic effect of D1 when combined with the other D suppressors, and the peculiar effect of D1 in combination with other Mhc alleles (Table III ), suggests that this suppressor elicits a different, albeit unknown, functional change. Direct interaction between the MHC regions identified by the other two suppressor mutations ( D41 and D45 ) and troponin I is not as obvious a possibility. However, it is important to realize that crystal structures of the myosin head represent static pictures of particular stages of the mechanochemical cycle. Thus, other contacts between thick and thin filaments are possible. A more likely explanation involves nucleotide exchange. Since both mutations are located near the nucleotide entry site of the molecule, it is reasonable to postulate that they would affect the ATPase cycle by regulating nucleotide entry or exit from the binding pocket . ADP release is the rate-limiting step in unloaded shortening of some muscles . If suppressor mutations reduce the rate of ADP release, myosin's dissociation from actin, which occurs upon subsequent binding of ATP, would be inhibited. This could dampen the unregulated actomyosin interactions that appear to occur in the hdp 2 mutant, since the ability of the myosin molecule to bind ATP and go through another step of the mechanochemical cycle would be reduced. Another consideration for the mechanism of suppression is that myosin could act through a third protein to regulate troponin I. In this situation, troponin I would interact indirectly with myosin, through another protein or protein complex (such as tropomyosin or other components of the troponin complex). When troponin I has an abnormal interaction with this partner in the hdp 2 mutant, the partner is unable to productively interact with myosin, unless a specific interacting site (the location of the suppressor mutation) is altered. Actin is an obvious possibility for such an intermediary protein, since it interacts with the troponin/tropomyosin complex, as well as with myosin. A key result of our study is that specific residues on MHC are required for suppression, suggesting they are critical to thick–thin filament interactions. None of the other alleles of Mhc , including point mutations, suppress the heldup wing phenotype (Table III ). This includes a mutation in the motor domain ( Mhc 5 ), a mutation in the lever arm ( Mhc 8 ), and a mutation in the rod ( Mhc 6 ). Interestingly, the genotype hdp 2 ; Mhc 5 /+ results in a lethal interaction . The location of this mutation close to the site of nucleotide entry/exit, and near D41 and D45 suppressors suggests that Mhc 5 might affect the ATPase cycle in the reverse direction of suppressors, thereby exacerbating rather than ameliorating the hdp 2 phenotypes. Support for this hypothesis is provided by the observation that lethality, but not heldup phenotype, of the hdp 2 ; Mhc 5 /+ genotype is eliminated when either the D41 , D45 , or D62 suppressors replace the wild-type Mhc allele (Table III ). D1 is an exception in rescuing lethality of the hdp 2 ; Mhc 5 combination. In contrast, MHC of the D1 type is compatible with Mhc 8 for viability, but this is not so with D41 , D45 , or D62 (Table III ). The opposite effects of D1 and other suppressor alleles strengthens our conclusion from suppressor heterozygote studies that D1 MHC acts to suppress the hdp 2 phenotype by a different mechanism than other suppressors. Our studies have implications for understanding disease processes in humans. In familial hypertrophic cardiomyopathy, single amino acid changes in a number of contractile proteins affect crossbridge cycling, resulting in myofibrillar disarray and hypertrophy . Mutations implicated in this disease include numerous defects in the myosin S1 domain and in troponin I . Thus, mutations in both thick and thin filament components can have similar consequences upon human cardiac muscle structure and function. A confounding factor in understanding the basis of disease process, and predicting its severity, is that genetic background influences disease penetrance. Our observations in Drosophila indicate that mutations in other components of the contractile apparatus can either exacerbate or ameliorate muscle dysfunction, and could serve as a model for understanding influences of genetic background upon disease penetrance. Further, our findings suggest suppression of human diseases by a mutated version of a contractile protein might prove useful in developing therapeutic strategies. | Study | biomedical | en | 0.999998 |
10085297 | Blot overlay was done using 35 S-labeled l-afadin, ponsin, or vinculin as described , with a slight modification. In brief, 35 S-labeled l-afadin, ponsin, and vinculin were generated with pBlue vectors containing full-length cDNAs of l-afadin, ponsin, and vinculin, respectively, using the TNT T7 quick coupled transcription/ translation system ( Promega Corp. ). The vinculin cDNA was kindly supplied by Drs. Y. Imamura, A. Nagafuchi, and Sh. Tsukita (Kyoto University, Kyoto, Japan). To remove free 35 S-methionine, each translated protein was applied to a Bio-Spin 6 chromatography column (Bio-Rad Laboratories). Each eluate from the column was used as a probe. The sample to be tested was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in PBS containing 5% defatted powder milk and 1% Triton X-100. The membrane was then incubated at 4°C for 16 h with the 35 S-labeled l-afadin, ponsin, or vinculin probe (∼2 × 10 6 cpm/ml each) in PBS containing 5% defatted powder milk and 1% Triton X-100. After the incubation, the membrane was washed with PBS containing 5% defatted powder milk and 1% Triton X-100, followed by autoradiography using an image analyzer . The AJ-enriched fraction was prepared from 80 rat livers as described , and stored at −80°C until use. Half of the AJ-enriched fraction was dissolved in an extraction solution (20 mM Tris-HCl at pH 9.0, 8 M urea, 1 mM DTT, and 1% hydrogenated Triton X-100; Calbiochem Corp. ). After sonication, the solution was mildly stirred at room temperature for 1 h and centrifuged at 100,000 g at 20°C for 30 min. The supernatant was diluted with a buffer (20 mM Tris-HCl at pH 9.0 and 1 mM DTT) to give final concentrations of 6 M urea and 0.75% hydrogenated Triton X-100. This sample (135 ml, 91 mg of protein) was applied to a Mono Q HR 10/10 column ( Amersham - Pharmacia Biotechnology ) equilibrated with buffer A (20 mM Tris-HCl at pH 9.0, 6 M urea, 0.75% CHAPS, and 1 mM DTT). After the column was washed with 70 ml of buffer A, elution was performed with a 160-ml linear gradient of sodium chloride (0–0.3 M) in buffer A. Fractions of 1.5 ml each were collected. When each fraction was subjected to 35 S-labeled l-afadin blot overlay, the radioactive bands appeared in fractions 14–25. These active fractions (18 ml, 3.1 mg of protein) were collected. The other half of the AJ-enriched fraction was also subjected to the Mono Q column chromatography in the same manner as described above. The active fractions of the two Mono Q column chromatographies were combined. The combined sample was applied to a hydroxyapatite column (0.75 × 7.5 cm; Tosoh) equilibrated with buffer B (8 mM potassium phosphate at pH 7.8, 6 M urea, 0.75% CHAPS, and 1 mM DTT). After the column was washed with 15 ml of the same buffer, elution was performed with a 25-ml linear gradient of potassium phosphate (8–100 mM) at pH 7.8 in buffer B. Fractions of 0.5 ml each were collected. When each fraction was subjected to 35 S-labeled l-afadin blot overlay, the radioactive bands appeared in fractions 25–33. The active fractions (4.5 ml, 1 mg of protein) were collected and applied to a Mono S PC 1.6/5 column ( Amersham - Pharmacia Biotechnology ) equilibrated with buffer C (50 mM sodium acetate at pH 4.0, 6 M urea, 0.75% CHAPS, and 1 mM DTT). After the column was washed with 0.7 ml of buffer C containing 0.15 M sodium chloride, elution was performed with a 2-ml linear gradient of sodium chloride (0.15–0.4 M) in buffer C. Fractions of 0.1 ml each were collected. When each fraction was subjected to 35 S-labeled l-afadin blot overlay, the radioactive bands appeared in fractions 9–13 . The active fractions were stored at −80°C. On the Mono S column chromatography, three radioactive bands with molecular masses of ∼95 (p95), 93 (p93), and 70 (p70) kD were identified by 35 S-labeled l-afadin blot overlay . The purified Mono S samples, fraction 10 for p93 and p95, and fractions 12 and 13 for p70, were subjected to SDS-PAGE (8% polyacrylamide gel). The protein bands corresponding to p95, p93, and p70 were separately cut out from the gel and digested with a lysyl endopeptidase, and the digested peptides were separated by TSKgel ODS-80Ts (4.6 × 150 mm; Tosoh) reverse phase HPLC as described . The amino acid (aa) sequences of the peptides were determined with a peptide sequencer. BLAST search of the GenBank database indicated that these sequences were almost identical to that of the mouse SH3P12 gene product . Based on the sequence of SH3P12 , oligonucleotide primers, 5′-CATTGGAAGACCTTGAGATCC-3′ and 5′-GAATGATGCTTCATCCTCCG-3′, were designed and a PCR reaction was performed using a mouse brain cDNA ( Clontech ) as a template. DNA sequencing was performed by the dideoxynucleotide termination method using a DNA sequencer (ABI 373). Prokaryote and eukaryote expression vectors were constructed in pCMV5 , pCMV-Myc , pFLAG-CMV2 ( Eastman Kodak Scientific Imaging Systems), pGEX ( Amersham - Pharmacia Biotechnology ), pMal-C2 ( New England Biolabs Inc. ), and pQE (QIAGEN Inc.) using standard molecular biology methods . pCMV-Myc and pFLAG-CMV2 were designed to express the proteins with the NH 2 -terminal Myc- and FLAG-epitopes, respectively. Various eukaryote expression constructs contained the following aa residues: pCMV5–l-afadin, 1–1829 (the full length); pCMV5–s-afadin, 1–1663 (the full length); pCMV-Myc–l-afadin, 1–1829 (the full length); pCMV-Myc–s-afadin, 1–1663 (the full length); pCMV-Myc–ponsin-1, 1–714 (the full length); pCMV-Myc–ponsin-2, 1–724 (the full length); pCMV-Myc–vinculin-N, 1–533 (the NH 2 -terminal half); pCMV-Myc–vinculin-C, 534–1066 (the COOH-terminal half); pCMV-Myc–vinculin-1, 1–800 (the NH 2 -terminal region); pCMV-Myc–vinculin-2, 801–1066 (the COOH-terminal region); pFLAG-CMV2–ponsin-1, 1–714 (the full length); and pFLAG-CMV2–ponsin-2, 1–724 (the full length). These constructs were transfected to COS7 cells with a DEAE-dextran method . The Myc-tagged recombinant proteins were purified from the cell extracts by immunoprecipitation with an anti–Myc antibody as described below. Various glutathione S -transferase (GST)–fusion constructs contained the following aa residues: GST–ponsin-2-F, 1–724 (the full length); GST–ponsin-2-N, 1–400 (the NH 2 -terminal half); GST–ponsin-2-C, 401–724 (the COOH-terminal half); GST–ponsin-2-SH3(1+2), 460–631 (the first and second SH3 domains); GST–ponsin-2-SH3(2+3), 543–724 (the second and third SH3 domains); GST–ponsin-2-SH3(1), 460–558 (the first SH3 domain); GST–ponsin-2-SH3(2), 543–631 (the second SH3 domain); GST–ponsin-2-SH3(3), 648–724 (the third SH3 domain); GST– vinculin-P, 837–878 (the proline-rich region); and GST–vinculin-C-ΔP, 879–1066 (the COOH-terminal region lacking the proline-rich region). Six histidine residues (His6)–fusion constructs contained the following aa residues: His6–l-afadin-C199, 1631–1829 (the COOH-terminal 199 aa region including the third proline-rich region); His6–l-afadin-C132, 1698–1829 [the COOH-terminal 132 aa region lacking the aa residues (PPLP) of the third proline-rich region]. A maltose-binding protein (MBP)–fusion construct (MBP–vinculin-C) contained the COOH-terminal half of vinculin . The GST-, His6-, and MBP-fused proteins were purified by use of glutathione-Sepharose beads ( Amersham - Pharmacia Biotechnology ), TALON metal affinity beads ( Clontech ), and amylose resin beads ( New England Biolabs Inc. ), respectively. Immunoprecipitation experiments from COS7 cells were done as follows: the eukaryote expression constructs described above were transfected to COS7 cells with the DEAE-dextran method in various combinations . A lysis buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (20 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM PMSF) was added to the transfected COS7 cells. The cell extract (1.2 mg protein), obtained by centrifugation at 100,000 g for 1 h, was incubated with either an anti–Myc antibody or an anti–FLAG M2 antibody (13.5 μg of protein; Eastman Kodak Scientific Imaging Systems) at 4°C for 3 h. 20 μl of protein G–Sepharose 4 Fast Flow beads ( Amersham - Pharmacia Biotechnology ) were added to this sample and incubation was performed at 4°C for 1 h. After the beads were washed with the lysis buffer, the bound proteins were eluted by boiling the beads in an SDS sample buffer for 10 min. The samples were then subjected to SDS-PAGE, followed by protein staining with Coomassie brilliant blue or by Western blot analysis. Affinity chromatography was done as follows: various GST-fused proteins of ponsin-2 and GST alone (200 pmol each) were separately immobilized on 10 μl of glutathione-Sepharose beads ( Amersham - Pharmacia Biotechnology ). His6–l-Afadin-C-199 (the COOH-terminal 199 aa region including the third proline-rich region), His6–l-afadin-C132 (the COOH-terminal 132 aa region lacking the aa residues [PPLP] of the third proline-rich region), MBP–vinculin-C (the COOH-terminal half), or MBP alone (200 pmol each) was incubated at 4°C overnight with the glutathione-Sepharose beads in 100 μl of PBS containing 0.1% Triton X-100. For competition experiments, His6–l-afadin-C199 (200 pmol or 2 nmol) and MBP-vinculin-C (200 pmol or 2 nmol) were mixed and incubated with 10 μl of the GST–ponsin-2-C (the COOH-terminal half)–immobilized beads in 400 μl of PBS containing 0.1% Triton X-100. The beads were extensively washed with the same buffer. Elution was performed with PBS containing 20 mM glutathione. A rabbit antiserum against ponsin was raised against GST–ponsin-2-SH3 (1) (the first SH3 domain). This antiserum was affinity-purified with GST– ponsin-2-SH3(1) covalently coupled to NHS-activated Sepharose beads ( Amersham - Pharmacia Biotechnology ) and used as a polyclonal antiponsin antibody. A mouse monoclonal anti–l-afadin antibody was prepared as described . A rabbit polyclonal anti–l- and s-afadin antibody, which recognized both l- and s-afadins, was prepared as described . A mouse monoclonal anti-vinculin antibody was purchased from Sigma Chemical Co. Rat monoclonal anti– E-cadherin (ECCD2) and anti–P-cadherin (PCD1) antibodies were purchased from TAKARA Shuzo, Inc. (Shiga). Immunofluorescence microscopy of cultured cells and frozen sections of various rat tissues was done as described . Immunoelectron microscopy using the silver-enhancement technique was done as described . 125 I-Labeled F-actin blot overlay and F-actin cosedimentation were done as described . Native vinculin was purified from chicken gizzard as described . COS7, mouse mammary tumor MTD-1A, and rat 3Y1 cells were maintained in DME containing 10% FCS. Protein concentrations were determined with BSA as a reference protein . SDS-PAGE was done as described . Fluorescence in situ hybridization (FISH) analysis was performed as described . To identify an l-afadin–binding protein(s), we used a blot overlay method with 35 S-labeled l-afadin. When each subcellular fraction of rat liver was subjected to SDS-PAGE, followed by the blot overlay, a radioactive band of ∼93 kD (p93) was detected and enriched in the AJ-enriched fraction . p93 was a major binding partner for l-afadin in the AJ-enriched fraction. p93 was solubilized from this fraction with a combination of urea and Triton X-100, and highly purified by successive chromatographies of Mono Q, hydroxyapatite, and Mono S columns. On the final Mono S column chromatography, the radioactive band with l-afadin–binding activity well coincided with a protein of ∼93 kD . p93 was purified with two other radioactive bands of ∼95 (p95) and 70 (p70) kD. They also coincided with proteins of ∼95 and 70 kD that were identified by protein staining with Coomassie brilliant blue. In addition to these proteins, a radioactive band of ∼55 kD (p55) was detected, but this band remained to be characterized. These protein bands were separately cut out from the gel and digested with a lysyl endopeptidase. The digested peptides were separated by HPLC, and peptide maps of these proteins were obtained. Their maps were similar to each other, but the maps of p95 and p93 had additional peptide peaks when compared with that of p70 (data not shown). When two peptide peaks of each of p95, p93, and p70, both of which were common among these proteins, were separately sequenced, each of the three proteins included the following peptide sequences: peptide-1, LNRDDDSDLHSPRYSFSEDTK; peptide-2, CDDGWFVGTSRRTK. BLAST search of the GenBank database indicated that these sequences were almost identical to that of the mouse SH3P12 gene product , which had originally been identified as an SH3-containing protein . These results suggest that p95, p93, and p70 are related to SH3P12. We performed PCR to amplify the cDNA of SH3P12 using a mouse brain cDNA as a template, and isolated at least 12 clones whose restriction maps were similar to but slightly different from each other (data not shown). We determined the nucleotide sequences of two of them, clones 2 and 17. Each clone contained an in-frame stop codon upstream of the predicted initiation codon. The protein encoded by clone 2 had 724 aa with a calculated molecular weight of 81,217 and the protein encoded by clone 17 had 714 aa with a calculated molecular weight of 79,492 . Both the proteins included the aa sequences identical to peptide-1 and -2, except that L (at the 9th aa residue) and E (at the 18th aa residue) of peptide-1 were replaced with V and D, respectively, and K (at the 14th aa residue) of peptide-2 was replaced with R. This difference in the aa residues is likely to be due to the species difference between mouse and rat. When the aa sequences of clones 2, 17, and SH3P12 were aligned, they were highly homologous (92% aa identity between clone 2 and SH3P12 and 96% aa identity between clone 17 and SH3P12) but their differences were clustered and not distributed throughout the sequences . To examine whether clones 2 and 17 were derived from the same locus, we made the chromosomal assignment by the FISH analysis using the cDNAs as probes. Clones 2 and 17 were located at the same locus (data not shown). These results suggest that clones 2, 17, and SH3P12 were splicing variants derived from the same gene. To examine whether the protein encoded by clone 2 or 17 was the mouse counterpart of p95, p93, or p70, we constructed a Myc-tagged mammalian expression vector with each DNA, and expressed the protein in COS7 cells. The Myc-tagged recombinant protein was immunoprecipitated with the anti–Myc antibody and subjected to SDS-PAGE, followed by 35 S-labeled l-afadin blot overlay. The protein encoded by clone 17 showed a mobility similar to that of native p93 on SDS-PAGE and the 35 S-labeled l-afadin– binding activity . On the basis of these observations, we concluded that clone 17 encoded the full-length cDNA of the mouse counterpart of p93 and named it ponsin-1, although we did not know the reason for the difference between the molecular mass value calculated from the predicted aa sequence and that estimated by SDS-PAGE. On the other hand, the protein encoded by clone 2 had the 35 S-labeled l-afadin–binding activity, but showed a mobility between p93 and p95 on SDS-PAGE. Thus, we could not conclude whether p95 was encoded by clone 2. The protein encoded by clone 2 was named ponsin-2. Ponsin-2 appeared to bind 35 S-labeled l-afadin more than ponsin-1 under the conditions used here. Hydrophilicity analysis of ponsin-1 and -2 predicted that these proteins had no transmembrane region (data not shown). Computer analysis of domain organization indicated that ponsin-1 and -2 had three SH3 domains at their COOH-terminal regions and two sorbin-like regions at their NH 2 -terminal regions, but did not have other known signaling domains . We confirmed the binding of l-afadin to ponsin by an immunoprecipitation method. Myc–l-afadin (the full length) and FLAG–ponsin (the full length) were transiently coexpressed in COS7 cells. When FLAG–ponsin-2 was immunoprecipitated with the anti–FLAG antibody, Myc– l-afadin was coprecipitated . Conversely, when Myc–l-afadin was immunoprecipitated with the anti–Myc antibody, FLAG–ponsin-2 was coprecipitated. Similarly, FLAG–ponsin-2 and tag-free l-afadin (the full length) or s-afadin (the full length) were transiently coexpressed in COS7 cells. When FLAG–ponsin-2 was immunoprecipitated with the anti–FLAG antibody, most of l-afadin was coprecipitated, but s-afadin was coprecipitated to a small extent and mostly recovered in the supernatant fraction . The essentially same results were obtained with ponsin-1 (data not shown). These results indicate that ponsin binds mainly l-afadin and slightly s-afadin. To examine the possibility that ponsin was an F-actin– binding protein and bound l-afadin through F-actin, we performed 125 I-labeled F-actin blot overlay and F-actin cosedimentation using the recombinant proteins as described . GST–ponsin-2-F (the full length) did not bind to F-actin under the conditions where l-afadin bound to F-actin (data not shown). We first determined the ponsin-binding region of l-afadin. 35 S-labeled ponsin-2 blot overlay analysis revealed that Myc–l-afadin (the full length), but not Myc–s-afadin (the full length) that lacked the third proline-rich region, bound to ponsin-2 . His6–l-Afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region), but not His6–l-afadin-C132 (the COOH-terminal 132 aa region lacking the aa residues [PPLP] of the third proline-rich region), bound to ponsin-2. These results suggest that the third proline-rich region of l-afadin binds to ponsin. We next determined the l-afadin–binding region of ponsin. 35 S-Labeled l-afadin blot overlay analysis revealed that GST–ponsin-2-F (the full length) and GST–ponsin-2-C (the COOH-terminal half) bound to l-afadin . In contrast, GST–ponsin-2-N (the NH 2 -terminal half) did not show this activity. In the COOH-terminal half of ponsin-2, GST–ponsin-2-SH3(2+3) (the second and third SH3 domains) showed this activity, but GST–ponsin-2-SH3 (1+2) (the first and second SH3 domains) did not. When each of the three SH3 domains was used, neither GST– ponsin-2-SH3(1) (the first SH3 domain), GST–ponsin-2-SH3 (2) (the second SH3 domain), nor GST–ponsin-2-SH3(3) (the third SH3 domain) bound to l-afadin. These results suggest that the region containing the second and third SH3 domains binds to l-afadin. We furthermore examined the binding regions of l-afadin and ponsin by an affinity chromatography method using the recombinant proteins. Consistent with the results by the blot overlay method, His6–l-afadin-C199 bound to GST–ponsin-2-C immobilized on glutathione-Sepharose beads, whereas His6–l-afadin-C132 did not . His6–l-afadin-C199 bound to GST–ponsin-2-F, GST–ponsin-2-C, and GST–ponsin-2-SH3(2+3), but not to GST– ponsin-2-N, GST–ponsin-2-SH3(1+2), GST–ponsin-2-SH3 (1), or GST–ponsin-2-SH3(2) . In contrast, His6–l-afadin-C199 slightly bound to GST–ponsin-2-SH3 (3). This inconsistency may be due to the sensitivity difference between the two methods. Because ponsin-1 and -2 were predicted not to be integral membrane proteins as described above, these proteins by themselves would not determine the specific localization of l-afadin at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells. To explore another molecule(s) through which l-afadin was localized there, we furthermore attempted to identify a ponsin-binding protein(s). To identify the protein(s), we performed a blot overlay method with 35 S-labeled ponsin-2. When each subcellular fraction of rat liver was subjected to SDS-PAGE, followed by the blot overlay, a radioactive band of ∼120 kD was detected and relatively enriched in the AJ-enriched fraction . Each fraction from the Mono Q column chromatography, which was used for the purification of ponsin, was subjected to 35 S-labeled ponsin-2 blot overlay. The radioactive band of ∼120 kD was detected in fractions 19–29 . The radioactive band well coincided with a protein band of ∼120 kD that was identified by protein staining with Coomassie brilliant blue (data not shown). This protein was recognized by the anti-vinculin antibody, suggesting that ponsin directly binds vinculin . We confirmed the binding of ponsin to vinculin by an immunoprecipitation method. Because vinculin shows the intramolecular association of its NH 2 - and COOH-terminal halves , we used the COOH- and NH 2 -terminal halves of vinculin. FLAG–ponsin-2 (the full length) and Myc–vinculin-C (the COOH-terminal half) were transiently coexpressed in COS7 cells. When FLAG–ponsin-2 was immunoprecipitated with the anti– FLAG antibody, Myc–vinculin-C was coprecipitated . Conversely, when Myc–vinculin-C was immunoprecipitated with the anti–Myc antibody, FLAG–ponsin-2 was coprecipitated. When Myc–vinculin-N (the NH 2 -terminal half) was used instead of Myc–vinculin-C, FLAG–ponsin-2 or Myc–vinculin-N was not coimmunoprecipitated. The essentially same results were obtained with ponsin-1 (data not shown). These results indicate that ponsin binds not only l-afadin but also vinculin both in cell-free and intact cell systems. We first examined the vinculin-binding region of ponsin. 35 S-labeled vinculin blot overlay analysis revealed that GST–ponsin-2-F (the full length) and GST–ponsin-2-C (the COOH-terminal half) bound to vinculin . In contrast, GST–ponsin-2-N (the NH 2 -terminal half) did not show this activity. In the COOH-terminal half of ponsin, GST–ponsin-2-SH3(1+2) (the first and second SH3 domains) bound to vinculin, whereas neither GST–ponsin-2-SH3(1) (the first SH3 domain) nor GST–ponsin-2-SH3 (3) (the third SH3 domain) bound. GST–ponsin-2-SH3 (2+3) (the second and third SH3 domains) and GST–ponsin-2-SH3(2) (the second SH3 domain) slightly bound to vinculin. These results suggest that the region containing the first and second SH3 domains binds to vinculin. We next examined the ponsin-binding region of vinculin. Consistent with the results by the immunoprecipitation method, 35 S-labeled ponsin-2 blot overlay analysis revealed that native vinculin and Myc–vinculin-2 , but not Myc–vinculin-1 (the NH 2 -terminal region, aa 1–800), bound to ponsin-2 . GST–vinculin-P (the proline-rich region), but not GST–vinculin-C-ΔP (the COOH-terminal region lacking the proline-rich region), bound to ponsin-2. These results suggest that the proline-rich region of vinculin binds to ponsin. The proline-rich region has two proline-rich sequences, one of which binds to the cytoskeletal protein, vasodilator-stimulated phosphoprotein . It remains to be clarified which proline-rich sequence of vinculin is responsible for the binding to ponsin. We furthermore examined the binding regions of ponsin and vinculin by an affinity chromatography method using the recombinant proteins. Consistent with the results by the immunoprecipitation and blot overlay methods, MBP– vinculin-C (the COOH-terminal half) bound to GST–ponsin-2-F, GST–ponsin-2-C, and GST–ponsin-2-SH3(1+2), but not to GST–ponsin-2-SH3(1) or GST–ponsin-2-SH3 (3) . MBP–vinculin-C slightly bound to GST– ponsin-2-SH3(2+3) and GST–ponsin-2-SH3(2). When MBP alone was used instead of MBP–vinculin-C, MBP alone did not bind to GST–ponsin-2-C . The above result that the l-afadin– and vinculin-binding regions of ponsin are partly overlapped suggests that l-afadin and vinculin bind to ponsin in a competitive manner and that these three proteins do not form a ternary complex. We examined this possibility by two different methods, immunoprecipitation and affinity chromatography. When FLAG–ponsin-2 (the full length) alone was transiently expressed in COS7 cells and immunoprecipitated with the anti–FLAG antibody, both endogenous l-afadin and vinculin were coprecipitated . However, it was not clear from this result whether a ternary complex of ponsin, l-afadin, and vinculin, or a mixture of binary complexes of ponsin and l-afadin and of ponsin and vinculin was coprecipitated. To address this issue, we examined whether ponsin and l-afadin or ponsin and vinculin were coprecipitated when vinculin or l-afadin was immunoprecipitated, respectively. Both FLAG–ponsin-2 and Myc– vinculin-C (the COOH-terminal half) were transiently coexpressed in COS7 cells. When Myc–vinculin-C was immunoprecipitated with the anti–Myc antibody, FLAG– ponsin-2, but not endogenous l-afadin, was coprecipitated . When FLAG–ponsin-2 was immunoprecipitated with the anti–FLAG antibody, both Myc–vinculin-C and endogenous l-afadin were coprecipitated, but the amount of the precipitated l-afadin was reduced, compared with that from COS7 cells expressing FLAG–ponsin-2 alone. Next, both FLAG–ponsin-2 and Myc–l-afadin (the full length) were transiently coexpressed in COS7 cells. When Myc–l-afadin was immunoprecipitated with the anti–Myc antibody, FLAG–ponsin-2, but not endogenous vinculin, was coprecipitated . When FLAG–ponsin-2 was immunoprecipitated with the anti– FLAG antibody, both Myc–l-afadin and endogenous vinculin were coprecipitated, but the amount of the precipitated vinculin was reduced compared with that from COS7 cells expressing FLAG–ponsin-2 alone. These results suggest that l-afadin and vinculin bind to ponsin in a competitive manner, and that l-afadin, ponsin, and vinculin hardly form a ternary complex. We confirmed this conclusion by an affinity chromatography method using the recombinant proteins. His6–l-Afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region) was mixed with MBP–vinculin-C (the COOH-terminal half) and incubated with GST–ponsin-2-C (the COOH-terminal half) immobilized on glutathione-Sepharose beads. When the concentration of His6–l-afadin-C199 was fixed and the concentration of MBP–vinculin-C was increased, the amount of the bound His6–l-afadin-C199 was decreased, whereas the amount of the bound MBP–vinculin-C was increased . Conversely, when the concentration of His6–l-afadin-C199 was increased and the concentration of MBP–vinculin-C was fixed, the amount of the bound His6–l-afadin-C199 was increased, whereas the amount of the bound MBP–vinculin-C was decreased. The exact affinities of l-afadin and vinculin to ponsin were not examined, but their affinities were apparently similar. We confirmed that l-afadin and vinculin did not bind to each other. Myc–l-afadin or Myc–vinculin-C alone was transiently expressed in COS7 cells. When Myc–l-afadin was immunoprecipitated with the anti–Myc antibody, endogenous vinculin was not precipitated . Similarly, when Myc–vinculin-C was immunoprecipitated with the anti–Myc antibody, endogenous l-afadin was not precipitated . In the next series of experiments, we analyzed the tissue distribution and intracellular localization of ponsin. Northern blot analysis using ponsin-2 as a probe showed that ponsin was ubiquitously expressed in all the mouse tissues examined, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis . In heart, brain, liver, and skeletal muscle, several bands were detected, consistent with the above result that ponsin had several splicing variants. Western blot analysis using the anti-ponsin antibody showed that ponsin as well as l-afadin was concentrated in the AJ-enriched fraction of liver . This antibody specifically recognized p93 (ponsin-1), p95, and p70. Vinculin was detected in all the fractions and relatively concentrated in the AJ-enriched fraction . To determine the intracellular localization of ponsin, we performed immunofluorescence microscopy of cultured cells and various rat tissues. At the junctional level of mouse mammary tumor MTD-1A cells, ponsin was colocalized with l-afadin and vinculin at cell–cell AJ . At the basal level, ponsin was colocalized with vinculin at cell–matrix AJ where l-afadin was not localized. In rat 3Y1 fibroblasts, ponsin was colocalized with l-afadin, vinculin, and P-cadherin at cell–cell AJ and with vinculin at cell– matrix AJ . When the frozen sections of rat small intestine were doubly stained with the monoclonal anti– E-cadherin antibody, ponsin was concentrated with E-cadherin at the junctional complex region of absorptive epithelial cells, but ponsin was more highly concentrated at the junctional complex region than E-cadherin was . When the frozen sections of rat heart were doubly stained with the monoclonal anti-vinculin antibody, ponsin was colocalized with vinculin at the intercalated disc. They were also periodically located along the lateral borders of cardiac muscle cells (costamere) . In liver, ponsin showed the beltlike localization along the bile canaliculi . This localization was consistent with its subcellular distribution in liver . These results indicate that ponsin is localized at both cell–cell and cell–matrix AJs, although l-afadin is localized only at cell–cell AJ, and that ponsin is colocalized with l-afadin at cell–cell AJ. To examine the precise localization of ponsin in the junctional complex region, we performed immunoelectron microscopy of rat small intestine absorptive epithelial cells using the silver enhancement technique. Ponsin was associated with the undercoat of ZA where l-afadin is localized . In this study, we isolated an l-afadin– and vinculin-binding protein, named ponsin, which was ubiquitously expressed and colocalized with vinculin at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells . We first purified three 35 S-l-afadin–binding proteins, p95, p93, and p70, from the AJ-enriched fraction of rat liver. We then analyzed the peptide maps and sequences of these proteins and found that they were related to each other. Based on their peptide sequences, we obtained the mouse cDNAs of ponsin-1 and -2. Ponsin-1 was likely a mouse counterpart of rat p93. However, ponsin-2 did not show the same mobility to that of p95 or p70 on SDS-PAGE. During the molecular cloning of ponsin, we obtained at least 12 independent clones, all of which had the similar restriction maps. Northern blot analysis indicated that ponsin had several mRNA bands in heart, brain, liver, and skeletal muscle. When the aa sequences of ponsin-1 and -2 were aligned together with that of SH3P12, they were highly homologous but their differences were clustered and not distributed throughout the sequences. Moreover, the FISH analysis indicated that ponsin-1 and -2 were located at the same locus (data not shown). We have determined neither the genetic locus of SH3P12 nor the clones other than ponsin-1 and -2, but ponsin/SH3P12 had many splicing variants, and all of these variants might be derived from the same gene. The exact relationship of the 35 S-l-afadin–binding proteins (p95 and p70) and ponsin-1 and -2 has not been clarified here, but there are two possibilities: (a) p95 and p70 are splicing variants of ponsin other than ponsin-1 and -2, and (b) they are posttranslationally modified forms of ponsin-1 or -2, such as the phosphorylated or proteolytic form. The physiological significance of the presence of many splicing variants of ponsin is unknown at present. We presented the evidence that l-afadin bound to ponsin by four different methods: (a) blot overlay, (b) affinity chromatography, (c) immunoprecipitation, and (d) immunofluorescence and immunoelectron microscopy. Blot overlay analysis indicated that ponsin-2 appeared to bind l-afadin more than ponsin-1, but their exact binding affinities remain to be clarified. We moreover showed that the binding regions of l-afadin and ponsin were the third proline-rich region and the region containing the second and third SH3 domains, respectively, consistent with the earlier observation that proline-rich sequences interact with either profilin or SH3 domains . Affinity chromatography analysis on each SH3 domain indicated that the third SH3 domain bound to l-afadin, whereas neither the first nor second SH3 domain bound, suggesting that the third SH3 domain is the minimal l-afadin–binding region. However, the l-afadin–binding activity of the third SH3 domain alone was less than that of the region containing the second and third SH3 domains. It remains to be clarified why the l-afadin–binding activity of ponsin is reduced when the second and third SH3 domains are separated from each other, but the highly ordered structure of the region containing the second and third SH3 domains may be necessary for the more efficient binding of ponsin to l-afadin. In contrast to l-afadin, s-afadin, which has the first and second proline-rich regions but lacks the third proline-rich region, hardly bound to ponsin. Neurabin-II, which also has three proline-rich regions , did not bind to ponsin either (data not shown). These results suggest that the SH3 domains of ponsin recognize a specific proline-rich region. Computer analysis indicated that ponsin-1 and -2 had three SH3 domains and two sorbin-like regions, but no transmembrane region or other known signaling domains. However, ponsin was localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells . These results suggest that there is another protein that determines the specific localization of l-afadin at these specific sites of cells. Along this line, we found here that ponsin-1 and -2 bound vinculin, which interacts with α-catenin at cell–cell AJ and with talin at cell–matrix AJ . α-Catenin interacts with cadherin through β-catenin , whereas talin directly interacts with integrin . We presented here the evidence that ponsin directly bound vinculin by four different methods: (a) blot overlay, (b) affinity chromatography, (c) immunoprecipitation, and (d) immunofluorescence and immunoelectron microscopy. Moreover, we showed that the binding regions of ponsin and vinculin were the region containing the first and second SH3 domains and the proline-rich region, respectively. The proline-rich region of vinculin is divided into at least two proline-rich sequences, the first of which binds to vasodilator-stimulated phosphoprotein (VASP) . We have not determined which proline-rich sequence is responsible for the binding to ponsin. Blot overlay and affinity chromatography analyses on each SH3 domain indicated that the second SH3 domain bound to vinculin, whereas neither the first nor third SH3 domain bound, suggesting that the second SH3 domain is the minimal vinculin-binding region. However, the vinculin-binding activities of the second SH3 domain alone and the region containing the second and third SH3 domains were less than that of the region containing the first and second SH3 domains. It remains to be clarified why the vinculin-binding activity of ponsin is reduced when the first and second SH3 domains are separated from each other. The highly ordered structure of the region containing the first and second SH3 domains may be necessary for the more efficient binding of ponsin to vinculin. The result (the l-afadin– and vinculin-binding regions of ponsin were partly overlapped) suggested that l-afadin and vinculin bind to ponsin in a competitive manner and that these three proteins do not form a ternary complex. Consistently, we found that l-afadin and vinculin bound to ponsin in a competitive manner and that the affinities of l-afadin and vinculin to ponsin were apparently similar. We found here by immunoprecipitation and affinity chromatography analyses that ponsin formed a binary complex with either l-afadin or vinculin, but hardly formed a ternary complex with l-afadin and vinculin. This result suggests that ponsin does not serve as a direct linker between l-afadin and vinculin. The physiological significance of the result that ponsin forms a binary complex with either l-afadin or vinculin at the same time but not a ternary complex with the two F-actin–binding proteins is currently unknown, but ponsin may independently regulate the function of each protein, or may regulate the linkage between l-afadin and vinculin in cooperation with another still unidentified factor. If the latter is the case, l-afadin may be localized at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells by interacting with the cadherin-catenin system through the ponsin-vinculin system . ZO and ZA in the junctional complex of polarized epithelial cells are closely aligned from the apical side to the basal side, suggesting that these two junction structures have their molecular interactions. ZO is also linked to the actin cytoskeleton. At ZO, multiple integral membrane proteins with four transmembrane regions, the claudin family members and occludin, constitute tight junction strands . The cytoplasmic region of occludin binds ZO-1 , which interacts with F-actin . The cytoplasmic regions of the claudin family members may also directly or indirectly bind ZO-1 . Evidence is accumulating that the cadherin-catenin system plays essential roles for the assembly of the junctional complex . It has recently been shown by use of an α-catenin–deficient colon carcinoma cell line that the binding of vinculin to α-catenin is required for the organization of ZO . It has furthermore been shown that the junctional organization is impaired in vinculin-null F9 cells . It should be noted from the present and previous results that l-afadin and ponsin as well as vinculin are more highly concentrated at ZA than cadherin. The afadin-ponsin system may be involved in the assembly of the junctional complex by interacting with vinculin. We recently found that l-afadin and E-cadherin showed different behavior during the formation and destruction of cell–cell AJ in MDCK and L cells . Dissociation of MDCK cells by culturing the cells at 2 μM Ca 2+ caused rapid endocytosis of E-cadherin, but not that of l-afadin or ZO-1. Addition of phorbol 12-myristate 13-acetate to these dissociated cells formed a ZO-like structure where ZO-1 and l-afadin, but not E-cadherin, accumulated. Even in cadherin-deficient L cells, l-afadin was mainly localized at cell–cell contacts, whereas ZO-1 was mainly localized at the tip area of cell processes. l-Afadin did not directly bind to α-, β-catenin, E-cadherin, ZO-1, or occludin. All of the results thus far available suggest that there is an integral membrane protein that is specifically localized at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells and interacts with the afadin–ponsin– vinculin system . It would be of crucial importance to identify this protein for our understanding of the physiological significance of l-afadin and ponsin. Ponsin-1 and -2 were splicing variants of SH3P12. SH3P12 was suddenly renamed CAP in the GenBank database during the revision of this manuscript. CAP was originally isolated as a c-Cbl–binding protein . c-Cbl is a proto-oncogene product involved in T cell antigen receptor–mediated signaling . In this CAP paper, no nucleotide or amino acid sequence information was available. Homology searches of DNA and protein databases furthermore revealed another protein structurally related to ponsin/ SH3P12, ArgBP2. ArgBP2 was isolated as an Arg- and Abl-binding protein . Arg and Abl represent the members of the Aberson family of tyrosine kinase. During the revision of this manuscript, Kioka et al. isolated a protein structurally related to ponsin/ SH3P12, named vinexin, as a vinculin-binding protein. All of these proteins have three SH3 domains and one or two sorbin-like regions. These results suggest that ponsin/ SH3P12/CAP, ArgBP2, and vinexin constitute a family. Structural comparison of these proteins suggest that ponsin/SH3P12/CAP, ArgBP2, and vinexin are derived from three different genes and constitute three subfamilies. Vinexin as well as ponsin/SH3P12 is localized at both cell– cell and cell–matrix AJs , whereas both SH3P12/CAP and ArgBP2 are associated with actin stress fibers . Vinexin enhances the actin cytoskeleton reorganization and cell spreading . SH3P12/CAP enhances actin stress fiber formation and focal adhesions and is associated with signaling molecules such as the insulin receptor, focal adhesion kinase (FAK), and SOS, a Ras small G protein exchanger . Thus, the members of the family that has three SH3 domains and one or two sorbin-like regions show similar and different subcellular localization and association with other proteins, suggesting their related but diverse functions. | Study | biomedical | en | 0.999997 |
10085298 | Mouse polyclonal anti–PSTPIP-2 antibody is described in Wu et al. . Rabbit polyclonal anti–PTP-PEST antibody is described in Charest et al. . Mouse monoclonal anticortactin is a gift from Dr. J. Thomas Parsons, University of Virginia, Charlottesville, VA. The following antibodies were purchased as indicated: rabbit anti–FAK (A17) from Santa Cruz Biotechnology, Inc. ; mouse antipaxillin from Transduction Laboratories; mouse antivinculin (hVIN-1) from Sigma Chemical Co. ; HRP-conjugated anti–mouse and –rabbit IgG antibodies and FITC-conjugated anti–mouse antibody from Jackson ImmunoResearch Laboratories, Inc. The antiphosphotyrosine antibodies used were mouse HRP-conjugated antiphosphotyrosine antibody (PY20) from Transduction Laboratories and the mouse monoclonal 4G10. To raise polyclonal antibodies against PSTPIP and p130 CAS , rabbits were injected intramuscularly with either 100 μg of purified GST PSTPIP (aa 247–415) or GST p130 CAS (aa 21–521) in PBS emulsified in complete Freund's adjuvant. Rabbits were boosted with the immunogens emulsified in incomplete Freund's adjuvant every 3 wk. Between each injection, samples of serums from rabbits were collected and analyzed for their abilities to detect either PSTPIP or p130 CAS in Western blotting experiments. When the titer of the antibodies in the serum was judged satisfactory, the antibodies were further purified by protein A–Agarose columns (Bio-Rad) for use in Western blotting or used directly for immunoprecipitation experiments (5 μl). The anti-PSTPIP antibody was conjugated using a peroxidase labeling kit ( Boehringer Mannheim Corp. ). The PTP-PEST (+/−) and (−/−) cell lines are described in Côté et al. . PTP-PEST (−/−), PTP-PEST (+/−), and COS-1 cells were maintained in DME supplemented with 10% FBS and penicillin/streptomycin. Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.). Subconfluent PTP-PEST (−/−) cells were transfected with this recombinant vector using Lipofectamine Plus ( GIBCO BRL ). Cells were first cultured in DME plus 10% serum for 48 h before selection with 25 μg/ml of Zeocin (Invitrogen Corp.). Zeocin-resistant clones were isolated and screened for expression of PTP-PEST by immunoblot analysis as described below. Exponentially growing COS-1 cells from a 15-cm tissue culture dish were transfected with 40 μg of pACTAG PTP-PEST by electroporation as described in Charest et al. . The cells were then grown for 48 h on Falcon chamber slides ( Becton Dickinson and Co.) in DME supplemented with 10% FBS and antibiotics. For EGF treatment, cells were serum starved by incubation in DME 0.1% FBS for 16 h followed by 10 min of treatment in 0.1% medium containing 100 ng/ml EGF (Upstate Biotechnology Inc.). For integrin activation, the cells were trypsinized and washed twice with DME containing 10% FBS and plated on fibronectin-coated slides (described above) for 45 min before fixing. Immunofluorescence was performed as described below, using 12CA5 (1:1,000) as a primary antibody. The capacity of each cell line to migrate on fibronectin was monitored by two different assays. In the wound healing assay, Falcon chamber slides were coated overnight at 4°C with a solution of fibronectin (10 μg/ml) in PBS, 10 mM sodium phosphate, 140 mM NaCl, pH 7.4. Cells were plated at 60% confluence in normal (10% serum) medium. After attachment, the monolayers were wounded by scoring with a sterile plastic 200 μl micropipette tip. Each well was then washed and fed daily with normal medium. After 24 h, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 5 min at room temperature and photographed using a low-magnification phase-contrast microscope. The extent of migration into the wound area was evaluated qualitatively. Transwell ® chambers (Corning-Costar) migration assays were performed as described in Klemke et al. . In brief, the under surface of the polycarbonate membrane (pore size, 8 μm) was precoated with fibronectin (10 μg/ml in PBS) overnight at 4°C. The membrane was washed to remove excess ligand and the lower chamber filled with DME containing 10% serum. PTP-PEST (+/−), PTP-PEST (−/−), and PTP-PEST (−/−) stably overexpressing PTP-PEST (10 5 in 100 μl of DME containing 10% serum) were added to the upper chamber, and left for 5 h at 37°C before fixing with 95% methanol in PBS. Unmigrated cells on the upper side of the membrane were removed with a cotton tip applicator, and the migrated cells were stained with methylene blue and counted using an inverted microscope (×40). Each determination represents the average of four individual wells and error bars represent SD. As a control, the lower side of the membrane was coated with 0.5% BSA. The background level of cell migration represented <0.1% of the individual experiments. Cells were plated on fibronectin-coated slides (described above) for 20 min or 3 h. They were then fixed 20 min with 4% (wt/vol) PFA in PBS, and permeabilized with 0.1% (vol/vol) Triton X-100 in 4% PFA for another 20 min. The slides were blocked with 1% (wt/vol) BSA in PBS for 20 min. The cells were then incubated with the antivinculin antibody (1: 400 dilution in PBS) for 1 h at room temperature, washed three times with PBS, and stained with a mixture of FITC-conjugated anti–mouse antibody (1:200) and 4 μl/well of TRITC-conjugated phalloidin (Molecular Probes Inc.). After being washed three times with PBS, the coverslips were mounted in a 1:1 mixture of glycerol and 2.5% 1,4-diazabicyclo [2.2.2]octane (DABCO; Sigma Chemical Co. ) in PBS. The cells were visualized with a Nikon fluorescence microscope. The number of focal adhesions in each cell was evaluated from photographs of the vinculin staining. For cleavage furrow staining, the cells were plated at low confluence on uncoated glass slides and left overnight in 10% serum containing medium. The cells were then fixed, permeabilized, blocked, and stained as described above. The primary antibody used was PY20. Dishes of cells were washed with PBS supplemented with 1 mM of sodium vanadate, harvested, lysed, and evaluated for protein content using the Bio-Rad protein assay. For immunoprecipitations of paxillin and cortactin, cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40). For FAK, PSTPIP, and PSTPIP2 immunoprecipitations, cells were lysed by sonication in HNMETG (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol). For vinculin and p130 CAS , the lysis buffer used was TNE buffer (10 mM Tris HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40). Lysis buffers were supplemented with 1 mM of sodium vanadate and Complete™ protease inhibitor mixture ( Boehringer Mannheim GmbH ). For each immunoprecipitation, 500 μg of total cell lysate was incubated with 1 μg of antibody and 30 μl of protein G–Agarose ( GIBCO BRL ) to a total volume of 1 ml in their respective lysis buffer (supplemented with vanadate and protease inhibitors) at 4°C for 90 min. The beads were then washed three times (the last wash for 15 min at 4°C) in their respective buffers, except FAK, PSTPIP, and PSTPIP2 which were washed in HNTG buffer (10 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol), and resuspended in 30 μl of SDS-PAGE loading dye. Western blots were performed following common procedures. Proteins were resolved on SDS-PAGE, transferred onto a PVDF membrane, blocked, and probed with the corresponding antibody in the same blocking buffer (1% BSA, 1% [vol/vol] goat serum, 0.1% Tween, in PBS). The following dilutions were used for the primary antibodies: 1:3,000 for 4G10 and anti–PTP-PEST; 1:10,000 for antipaxillin; 1:1,000 for antivinculin, anti-p130 CAS , anticortactin, and the HRP-conjugated pY20; 1:100 for anti-FAK. Secondary antibodies were used at 1:10,000. The Renaissance™ chemiluminescence kit ( New England Nuclear Life Science Products) was used for detection. 3.5-cm tissue culture dishes were coated with 1 μg/cm 2 of fibronectin overnight at 4°C. Plates were washed with PBS, and 1 ml of normal medium was prewarmed in a humidified 37°C incubator. Cells were trypsinized, counted, and 5 × 10 5 cells were added with medium to the plates for a final volume of 2 ml. Random fields were photographed after 10, 15, and 30 min using a low magnification phase-contrast microscope. Photographs were evaluated for the percentage of spread cells. Unspread cells were described as phase-bright and punctual, whereas spread cells were not phase-bright, with extensive visible membrane protrusions. The two kinds of cells were distinguishable enough so that two independent counts of the same field gave the same result ±2%. Four fields were counted for each cell line and, in each field, >300 cells were counted. The experiment was repeated four times. In vitro binding studies using glutathione S-transferase (GST) fusion proteins were performed as described elsewhere . In brief, 0.5 mg of each cell lysate was precleared with GST bound to glutathione–Sepharose beads ( Pharmacia Biotech, Inc. ) for 1 h at 4°C. The supernatants were then incubated with the GST-fusion protein prebound to glutathione–Sepharose beads in a total of 1 ml of TNE buffer supplemented with 0.1 mM vanadate for 90 min at 4°C, washed three times with TNE buffer, and resuspended in an equal volume of 2× SDS-PAGE loading buffer. Protein complexes were separated by SDS-PAGE and detected by immunoblotting as described above. The GST-Grb2 FL construct is described in Charest et al. . The GST-SH2 domains of Crk and Src are a generous gift from Dr. Park (McGill University). Because p130 CAS is known to translocate to focal adhesions when phosphorylated by the integrin pathway , and since PTP-PEST and p130 CAS associate via an SH3 proline-rich domain interaction , we first verified if PTP-PEST could translocate within a cell following integrin stimulation. COS-1 cells were transfected with a PTP-PEST construct tagged with the hemagglutinin antigen (HA) epitope, allowing visualization by indirect immunofluorescence . Untransfected cells showed no immunofluorescence staining , demonstrating the absence of nonspecific staining with the 12CA5 antibody. In transfected cells , PTP-PEST localization was diffuse in the cytoplasm, as previously described . When the cells were stimulated with 0.1 μg/ml of EGF, no significant change in PTP-PEST staining was observed , although PTP-PEST was shown previously to associate with the EGF receptor via Grb2 . However, for a significant percentage of attached transfected cells plated on fibronectin-coated slides for 45 min, staining for the HA epitope showed a clear relocalization to the membrane periphery even with lower levels of protein expression . These results prove that, in COS-1 cells, PTP-PEST can relocalize to the membrane periphery following integrin, but not EGF receptor, stimulation. Two recent reports suggested a correlation between p130 CAS phosphorylation level and cell migration rate. In both studies, a kinase was transfected in the cells. One used v-Src, for which p130 CAS is a direct substrate , while the other overexpressed FAK, that, once activated, binds both c-Src and its substrate p130 CAS . In both studies, hyperphosphorylation of p130 CAS was associated with an increase in rate of migration. Since p130 CAS is a substrate for PTP-PEST , removal of this PTP results in hyperphosphorylation of p130 CAS . We decided to investigate whether the absence of this PTP would result in a change in the motility of these cells. The two cell lines used were heterozygous and homozygous for the PTP-PEST deletion. To minimize any dominant negative effects from the targeted allele, comparisons were made between the homozygous and the heterozygous cell lines. One of the simplest assays to qualitatively compare cell migration is the wound healing migration assay. Fibroblast monolayers plated on fibronectin-coated slides were wounded at 37°C and fixed after 24 h. Fig. 2 shows a typical region of the wound for each cell line obtained after five independent experiments. The PTP-PEST (+/−) cells were able to migrate into the wound at a rate greater than the front of cells pushed in by proliferation , whereas the (−/−) cells showed a complete absence of chemokinetism and were not able to actively invade the wounded area. A more quantitative assay for migration is the chamber mobility assay, where the ability of cells to translocate to the fibronectin-coated side of a perforated membrane is measured (see Materials and Methods). Once again, we compared the migration of PTP-PEST (+/−) and PTP-PEST (−/−) cells. As in the wound-healing assay, a significant decrease in motility was observed in the knockout cells . To ensure that this effect is indeed due to the absence of PTP-PEST, PTP-PEST (−/−) cells were transfected with PTP-PEST and a stably overexpressing clone was obtained. When tested in the same assay, these cells showed an increase in migration that was significant but still not comparable with wild-type levels. One possibility is that overexpression of PTP-PEST could also have deleterious effects on cell migration, as is the case with PTP1B . To test this hypothesis, we are currently trying to obtain clones expressing more physiological levels of PTP-PEST, as well as different constructs and catalytically inactive mutants of the phosphatase, and verify the effects on this phenotype and the others described below. The two migration assays are complementary, since each has different limitations. For instance, the wound healing assay is affected by the rates of cell proliferation and by cell–cell adhesive interactions which restrict the ability of the cells to move into the wounded area. In the chamber assay, differences in cell shape and size may alter the ability of the cells to pass through the pores. Taken together, the data suggest that the loss of PTP-PEST results in motility defects in embryonic fibroblast cell lines that can be reincreased by overexpression of PTP-PEST in the (−/−) cells. The cytoskeleton of the cell plays an important role in cell motility. We investigated whether this observed migration impairment of homozygous PTP-PEST mutant cells could be caused by an abnormal organization of the actin filaments, or by a visible difference in focal adhesions. Cells from each cell line were plated on fibronectin. The actin filaments were stained with a rhodamine-conjugated phalloidin , and the focal adhesions were highlighted by indirect immunofluorescence using mouse mAb against vinculin . 25 min after plating, both cell lines showed membrane ruffles and filopodia , suggesting that the defect in migration was not caused by an incapacity to polymerize actin or to organize these structures. Large, immature focal adhesions were also found in each cell line , showing that the initial pathways forming these contacts were intact in PTP-PEST (−/−) cells. However, when cells were left for 3 h on fibronectin before fixing and staining, the homozygous cells remained well spread, with numerous focal adhesions scattered throughout the ventral surface of the cell . Surrounding the cells were small, hair-like actin fibers that characterize cells in the early stages of apolar migration . The PTP-PEST (+/−) cells, in contrast, were rounded, with well-defined edges and fewer focal contacts concentrated at the cell periphery . The (−/−) cells also contained a higher number of stress fibers, a state that is incompatible with cell migration. A quantitative evaluation of the number of focal adhesions after 3 h, as counted on vinculin-staining photographs, is shown in Fig. 3 i. PTP-PEST (−/−) cells have, on average, >85 focal contacts per cell, compared with ∼25 in a (+/−) cell. Thus, the migration defect observed in the wound healing assay above may be in part due to disregulation of focal adhesion assembly and/or disassembly in the PTP-PEST cell lines. Attempting to understand the differences observed in the size and number of focal adhesions between the two cell lines which differ only by the presence or absence of PTP-PEST, we analyzed the tyrosine phosphorylation state of specific focal adhesion proteins . The adapter protein p130 CAS was previously shown to be hyperphosphorylated , since it is a physiological substrate for PTP-PEST . The other proteins immunoprecipitated and analyzed for their phosphotyrosine status with an antiphosphotyrosine antibody included paxillin and cortactin. Paxillin was shown to be hyperphosphorylated in FAK null cell lines, a mutation that was also associated with a decrease in cell mobility and increase in numbers of focal adhesions . Paxillin phosphorylation was also shown recently to be required for cell spreading and focal adhesion formation . In the present study, paxillin was found in a hyperphosphorylated state . Interestingly, paxillin was shown to physically associate with PTP-PEST , but experiments using trapping mutants suggest that paxillin is not a direct substrate for PTP-PEST (Côté, J.-F., C.E. Turner, and M.L. Tremblay, manuscript submitted for publication). Cortactin, like p130 CAS , is a pp60Src substrate , although it is not associated with focal adhesions. It interacts with the actin cytoskeleton and is tyrosine phosphorylated following integrin-mediated cell adhesion to extracellular matrix . However, Fig. 4 a shows that cortactin is not constitutively tyrosine hyperphosphorylated in the PTP-PEST (−/−) cells, suggesting that it is not a direct or indirect substrate for this phosphatase. Vinculin is a structural protein that links talin and actin when focal adhesions form. It can be tyrosine phosphorylated, but this phosphorylation does not seem to be implicated in focal adhesion formation, the latter due to a conformational change in vinculin following phosphatidyl inositol biphosphate binding . There was no detectable basal level of tyrosine phosphorylation of vinculin in both cell lines, potentially due to the fact that the cells were not stimulated. Another focal adhesion component to be investigated was FAK. The role of p130 CAS in migration was shown to reside in the pathways triggered by FAK , which is associated with the integrins and becomes active by transphosphorylation when they cluster. Phosphorylation of Y397 on FAK, which is the binding site of the Src kinase, was shown to be crucial for its role in cell migration . Fig. 4 a shows that FAK is slightly hyperphosphorylated in the PTP-PEST null cells, suggesting that this PTP may regulate its phosphorylation level. Since FAK and PTP-PEST were never shown to interact directly, it is possible that this hyperphosphorylation is a consequence of p130 CAS or paxillin hyperphosphorylation. Also, to follow our results showing that p130 CAS is constitutively hyperphosphorylated in PTP-PEST (−/−) cells , we verified if this hyperphosphorylation was decreased in the clone overexpressing wild-type PTP-PEST used in Fig. 2 e. p130 CAS was immunoprecipitated from lysates of PTP-PEST (+/−), (−/−), and (−/−) overpressing PTP-PEST. The phosphorylation levels of p130 CAS in each cell line were then evaluated as for the focal adhesion proteins described above. Fig. 4 b shows that the phosphorylation of p130 CAS is decreased to normal levels in the PTP-PEST (−/−) cells overexpressing PTP-PEST. Taken together, these data suggest that PTP-PEST mediates the dephosphorylation of a specific subset of focal adhesion–associated proteins. This hyperphosphorylation of p130 CAS , paxillin, and FAK is constitutive, meaning the cells were not stimulated with growth factors or extracellular ligands. Also, cell spreading was associated with paxillin tyrosine phosphorylation . Finally, overexpression of PTP1B, a PTP for which p130 CAS is a substrate , caused inhibition of cell spreading . These facts led us to investigate whether the PTP-PEST (−/−) cells could be, on the contrary, more prone to attach on fibronectin since p130 CAS , paxillin, and FAK are already hyperphosphorylated. Normally growing cells were trypsinized and plated on fibronectin-coated tissue culture plates. After 10, 15, and 30 min, random fields were photographed and the cells were counted for the extent of cell spreading. The results of two independent experiments with different populations of the two same PTP-PEST (−/−) and (+/−) clones that were tested in the preceding experiments are shown in Fig. 5 . After 10 min, more PTP-PEST (−/−) are opaque, which is a characteristic of spread cells under phase-contrast microscopy . Photographs of fields after 30 min as well as a quantitative curve of the spreading time course are also shown. These results suggest that the PTP-PEST (−/−) cells are primed for attachment and spreading via integrin-stimulated pathways, and that PTP-PEST has a physiological role in regulating this event. The negative role of PTP-PEST in cell spreading is consistent with its role described above in cell migration via the breakdown of cell–substratum links. To understand where the tyrosine residues hyperphosphorylated on p130 CAS , paxillin, and FAK in the PTP-PEST ( − / − ) cells are, and to verify if these sites could be specific SH2-binding motifs, we examined the physical association of each of these proteins with a panel of SH2 domains in vitro. The three constructs tested were the GST fusion proteins of the Src and Crk SH2 domains, as well as the full-length Grb2 . These three SH2 domains were shown to bind phosphotyrosines on FAK , Src and Crk to p130 CAS , and Crk to paxillin . Each cell lysate was first precleared with GST alone to eliminate nonspecific binding (see Materials and Methods). 0.5 mg of the remaining proteins was then incubated with each GST-fusion protein and the bound proteins were resolved by SDS-PAGE . Between the PTP-PEST (+/−) and (−/−) cell lines, the greatest difference in affinity is observed between the SH2 domain of Src and p130 CAS and, to a lesser extent, between the SH2 domain of Crk and p130 CAS , strongly suggesting that the tyrosines within these SH2-binding domains on p130 CAS are hyperphosphorylated. The other lanes show a small and fairly constant (∼50%) increase in affinity, that may or may not be indirect and could reflect a general constitutive increase in focal complexes in the PTP-PEST (−/−) cells. The exact tyrosines that are hyperphosphorylated on paxillin and FAK are still under investigation. They may not be found in putative SH2-binding domains, but can play other roles in focal adhesion turnover. While we investigated the effects of PTP-PEST targeting in cell migration, another protein involved with the actin cytoskeleton, PSTPIP, was cloned and shown to be a substrate for a PTP of the PEST family, PTP-HSCF . A putative coiled-coil region of PSTPIP interacts with the COOH-terminal, proline-rich region of PTP-HSCF , a region that is conserved in all members of the PEST family, including PTP-PEST. Using a polyclonal antibody raised against PSTPIP, we analyzed its tyrosine phosphorylation level in the PTP-PEST (−/−) and (+/−) cells by immunoprecipitation, followed by antiphosphotyrosine immunoblotting. Data from Spencer et al. suggested that PSTPIP tyrosine phosphorylation was controlled by the members of the PEST family of PTPs, and Fig. 7 shows that PSTPIP was hyperphosphorylated in the PTP-PEST knockout cell line. Taken together with the fact that PSTPIP is a direct substrate for PTP-HSCF , and that this substrate effect required the conserved COOH-terminal proline-rich binding domain, these results suggest that PSTPIP could be a substrate for PTP-PEST in fibroblasts. The same experiment was performed on PSTPIP2, a protein with high homology to PSTPIP which binds to the PEST type PTPs but does not contain the SH3 domain, and is not a substrate for the PEST family of PTPs . The phosphorylation level of PSTPIP2 was similar in the (+/−) and the (−/−) cell lines (data not shown), suggesting the presence of another level of specificity, such as substrate recognition/activity in PTP-PEST. PSTPIP is homologous to S. pombe CDC15, an actin-associated protein that is critically involved in the formation of the cleavage furrow during cell division . During our manipulations of the PTP-PEST (−/−) cells, we observed a high occurrence of cells that seemed blocked in a late stage of cytokinesis, with the two daughter cells almost independent, but still attached to each other by an actin-rich junction which appears to be derived from the cleavage furrow. Fig. 8 a shows the actin cleavage furrow rings of several of these cells as stained by rhodamine-phalloidin. Fig. 8 b represents the same cells showing the indirect antiphosphotyrosine immunostaining and revealing a clear band of tyrosine phosphorylated material at the cleavage furrow. To eliminate the possibility of contamination of the rhodamine fluorescence through the FITC filter, the same antiphosphotyrosine staining was performed in the absence of rhodamine-phalloidin, with the same result. These cells were found in all PTP-PEST (−/−) cultures that we observed, and were virtually absent from any other wild-type cell lines. The rare PTP-PEST (+/−) cells found in this phase were also probed for antiphosphotyrosine and the cleavage furrow did reveal some staining. Fig. 8 b does not represent a phenotype but shows the implication of tyrosine phosphorylation in cytokinesis, which can explain why a PTP could play a role in this event. This phenomenon was postulated to be the result of a longer M phase due to an impairment of the actin rearrangement during division, a process where tyrosine phosphorylation is involved. FAK activation and focal adhesions formation are closely related events. Until recently, the exact order in which they occur following integrin stimulation was greatly debated. Recent experiments showed that FAK activation is a result of focal adhesion formation. Due to the physical tension caused by the stress fiber formation in a cell, under the control of the Rho GTPase, the associated integrins cluster and associate with other structural proteins like α-actinin and tensin . FAK proteins are associated with integrins and can transphosphorylate in a manner similar to receptor tyrosine kinases following extracellular ligand binding. This phosphorylation activates FAK and provides docking sites for other signal transduction proteins that propagate integrin-triggered pathways. However, the relationship between focal adhesion formation and FAK activation is apparently not strictly linear. Among the signals sent by FAK, some were shown to feed back and play a role in the turnover rate of focal adhesions. The existence of such signals was concluded when the FAK null cell lines showed an increase in focal adhesion size and number . However, these results did not take into account the effects of other members of the FAK family that were later identified, such as Pyk2 . Recently, FAK was shown to play a role in migration, an event that requires focal adhesion formation and breakdown. This increase in migration occurs via the tyrosine phosphorylation of an adapter protein, p130 CAS . Also, gene disruption of a cytoplasmic tyrosine phosphatase with two SH2 domains, SHP-2, was shown to impair the ability of fibroblasts to spread and migrate on fibronectin . This phenotype was associated with a decrease in FAK dephosphorylation following cell detachment and suspension. In this paper, we examined another cell line with a gene inactivated by homologous recombination, cytosolic PTP-PEST. A major substrate for this enzyme in both mice and humans was shown to be p130 CAS , and we show in Fig. 1 that, in COS-1 cells, PTP-PEST can translocate to the membrane periphery following integrin activation, possibly in order to reach the p130 CAS substrate which also translocates to focal adhesions when FAK is activated . The effects of PTP-PEST removal on cell migration and focal adhesion number were similar to the phenotype of the FAK mutant cells . Both showed a decrease in fibroblast migration using fibronectin as an extracellular matrix, and the presence of numerous immature focal adhesions scattered on the ventral face of the cell . Even though FAK and PTP-PEST have opposite catalytic activities on a common substrate, p130 CAS , PTP-PEST does not seem to antagonize the effects of FAK in cell migration, but rather to potentiate these effects. This suggests a mechanism where both a formation and a breakdown pathway are triggered at the same time to increase the turnover rate of the focal adhesion structures. Without the pathway required for focal adhesion breakdown, the adhesive contacts between the extracellular matrix and the cell are too strong, and the cell adheres and does not migrate . In FAK (−/−) cells, the actin stress fibers were dense around the cell periphery rather than in the center, where they can exert the transcellular tension necessary for cell migration . In contrast, in the PTP-PEST (−/−) cells, central stress fibers are omnipresent, suggesting that the cells cannot turn them over. This state is also incompatible with cell migration. The absence of differences in the early stages of cell attachment to fibronectin suggests that PTP-PEST is not implicated in the initial formation of stress fibers or focal adhesions. However, at equilibrium, clear differences appear . The focal adhesions in the PTP-PEST (−/−) cells are numerous and scattered throughout the ventral (i.e., substrate-attached) membrane of the cell, which can in part explain the absence of motility of the cells when tested in the wound healing assay. In contrast, the heterozygous cells became rounded, their focal adhesions were punctate and found only at the tips of their membranes, which is consistent with a migrating cell. This led us to believe that PTP-PEST is implicated in focal adhesion and stress fiber breakdown, a role that is the counterpart of FAK or Src but is required for the successful achievement of the same event, cell migration. In the PTP-PEST mutant cell line, p130 CAS , paxillin, and FAK were found in a hyperphosphorylated state. The phosphorylation of paxillin was shown to be associated with cell spreading on an extracellular matrix , which is consistent with data reported here. Paxillin can associate with PTP-PEST , but it does not constitute a direct physiological substrate for this PTP (Côté, J.-F., C.E. Turner, and M.L. Tremblay, manuscript submitted for publication) and the exact site that is hyperphosphorylated is still not clear. SH2-domain affinity assays shown in Fig. 4 strongly suggest the sites that are hyperphosphorylated on p130 CAS are within the SH2-binding domains of Src and Crk. One hypothesis is that the only tyrosine that is substrate for PTP-PEST is the 762YDYV765 Src SH2-binding region on p130 CAS , which binds Src in a tyrosine-dependant manner . In PTP-PEST (−/−) cells, this can cause Src to constitutively bind p130 CAS and hyperphosphorylate the Crk-binding motifs on p130 CAS . This would explain why p130 CAS is so hyperphosphorylated in the PTP-PEST (−/−) cells, and why the difference in affinity is greater with the Src SH2 domain compared with the Crk SH2 domain, even though there are 15 putative binding domains for Crk compared with only one for Src on p130 CAS . In the same line of thought, this could also be the cause for the small FAK hyperphosphorylation in the PTP-PEST (−/−) cells, since the SH3 domain of p130 CAS can bind a proline-rich region on FAK , and FAK is also a substrate for Src . This increased affinity between Crk and p130 CAS in the PTP-PEST (−/−) cells can be related to the phenotypes observed in Figs. 2 and 3 . The CAS/Crk coupling has been shown to play a role in the induction of cell migration . Thus, incorrect regulation of this molecular switch in the absence of PTP-PEST could lead to aberrant cell migration. These results provide a concrete, tyrosine phosphorylation–dependent way for PTP-PEST to regulate cell migration, and strongly suggest the phenotypes observed here are the result of the gene targeting. Still, reintroduction at physiological levels of different PTP-PEST constructs in the knockout cells to differentially rescue the phenotypes described in this paper is currently under way in our laboratory. Studies using the PTP inhibitor phenylarsine oxide showed that treatment of cells with this compound was sufficient to induce the formation of stress fibers even after starving them for 16 h . Phenylarsine oxide reacts with two thiol groups of closely spaced cysteine residues in the active site of the phosphatase. The PTP-PEST catalytic domain contains the sequence 231CSAGC235 , the cysteine at position 231 being crucial for its catalytic activity. These studies also showed that focal adhesion disassembly results in stimulation of phosphatase activity, which could be assayed using FAK and paxillin as substrates. That, and the fact that paxillin is hyperphosphorylated in the PTP-PEST knockout cell line, suggest that PTP-PEST is a candidate PTP involved in the focal adhesion breakdown in the conditions studied in these experiments. The role of a PTP in focal adhesion breakdown would suggest that overexpression of the PTP would also inhibit cell migration, by impairing the formation of the focal adhesions at the leading edge of the cell. Experiments involving another PTP that can dephosphorylate p130 CAS , PTP1B , showed that overexpression of this PTP in rat fibroblasts decreased cell migration while increasing the time required for the cell to spread on fibronectin . This was linked to a disordered formation of focal adhesions. In this article, we demonstrated that removal of a PTP that can dephosphorylate p130 CAS , PTP-PEST, increased the spreading speed of targeted cells on the same extracellular matrix protein . Interestingly, the PTP1B-overexpressing cells eventually formed numerous, large focal complexes scattered over their ventral surface, like ones found in the PTP-PEST (−/−) cells. These experiments and the ones presented in this paper suggest that an intermediate level of PTP activity towards p130 CAS is required for the formation of normal focal adhesions and for cell migration, which is consistent with a role in focal adhesion turnover. PTP-PEST may also play a role in the regulation of the cell cytoskeleton, this time via the cleavage furrow–associated protein PSTPIP. PSTPIP was originally identified as a binding partner and a substrate for the phosphatase PTP-HSCF , a PEST tyrosine phosphatase. PTP-HSCF dephosphorylates tyrosine residues in PSTPIP that are modified either by coexpression of the v-Src tyrosine kinase or in the presence of the unspecific PTP inhibitor pervanadate. One of these sites, within the SH3 domain of PSTPIP, was shown to regulate binding with the proline-rich region found on WASP , and to control aspects of the actin cytoskeleton. The possibility that tyrosine phosphorylation is involved in furrow development and the signaling events coordinating nuclear division was reported by Cool et al. . Their data show that BHK cells overexpressing a truncated PTP, namely T cell PTP, become highly multinucleate, apparently through a failure in cytokinesis. This deletion mutant lacked the COOH-terminal extension responsible for its proper localization, and the redistribution of the enzyme to the soluble fraction caused both a furrowing defect and an asynchronous entry into S phase of two nuclei within the same syncytial cell. It has not been shown whether PTP-PEST binds directly to PSTPIP, but peptides derived from the conserved proline-rich COOH terminus of PTP-PEST can compete the binding of PTP-HSCF to PSTPIP , consistent with the suggestion that PTP-PEST binds to PSTPIP in a manner similar to PTP-HSCF binding. In addition, while no previous data had shown that PTP-PEST can dephosphorylate PSTPIP, the fact that PSTPIP is hyperphosphorylated in the PTP-PEST (−/−) cells strongly suggests that PTP-PEST plays a direct role in modulating the tyrosine phosphorylation level of PSTPIP. PTP-HSCF and another member of the PEST family, PTP-PEP, are generally not expressed in fibroblasts, and it is possible that PTP-PEST regulates PSTPIP in fibroblasts the way PTP-HSCF does in hematopoietic cells. The exact site that is hyperphosphorylated on PSTPIP in PTP-PEST (−/−) cells is not known, and the effects of this hyperphosphorylation on binding, for example, to WASP are still under investigation. One of the observable effects appears to be an increase in the relative amount of cells found in the last stages of cytokinesis in a field of unsynchronized cells , possibly due to the role of PSTPIP in cleavage furrow assembly or disassembly. It is also possible that the increase of PSTPIP tyrosine phosphorylation is the result, and not the cause, of the increase of the number of cells in this phase. The fact that PTP-PEST (−/−) cells still divide and grow at rates comparable to other cells suggests the presence of other mechanisms involved in cell division. Isolation and characterization of cytokinesis-deficient mutants in Dictyostelium discoideum , a highly motile organism that undergoes cell cleavage much like higher eukaryotes, provided examples of cell division occurring with impaired cytokinesis . In particular, a mutation in the myosin gene that prevents the protein assembly in thick filaments resulted in organisms clearly defective in the contractile events involved in cytokinesis . Another mutant, called 10BH2, also had a complete defect in cytokinesis . However, both mutants, which showed cell division defects when grown in suspension, were able to divide when plated on solid substratum by pinching off a portion of their cytoplasm in a process known as traction-mediated cytofission . It is possible that the PTP-PEST (−/−) cells in part rely on this event to normally grow, even if the defect in chemokinesis observed in the wound-healing assay could impair the required migration. Interestingly, another D. discoideum mutant, in the single gene encoding calmodulin, formed and constricted a contractile cleavage furrow ring, but the midbody linking the daughter cells failed to completely close . The resultant population contained cells resembling cells observed in the PTP-PEST (−/−) population. This cytoplasmic bridge could be broken by shear forces when the cells were grown in suspension cultures and the cells could multiply normally. This suggests that cells in this state require little force to successfully complete division, and could explain the fact that the PTP-PEST (−/−) cells can still divide. In this article, we reported two roles that a specific PTP, PTP-PEST, could play in regulating the cytoskeleton of fibroblasts. The first, possibly via its capacity to dephosphorylate p130 CAS , is to break down focal adhesions, an event which is required for cell migration on an extracellular matrix like fibronectin. PTP-PEST was shown to localize at the membrane periphery when COS-1 cells were plated on fibronectin and this confers to PTP-PEST a physiological role in cell migration that is not a secondary effect of overexpression. The fact that PTP-PEST is mostly found in a cytoplasmic pool and can be recruited to the plasma membrane after fibronectin-mediated attachment provides the cell a mechanism to increase its focal adhesions turnover rate proportional to the stimulus, even when FAK, p130 CAS , or v-Src is overexpressed . PTP-PEST also plays a role in modulating the phosphorylation level of PSTPIP, a protein that associates with the cytoskeleton . The role of this PTP-PEST activity is not known, but it may involve the binding of WASP to PSTPIP . Since WASP has been shown to regulate actin fiber assembly and cytokinesis in both yeast and mammalian cells. These data suggest that this interaction may be somehow modulated in the PTP-PEST (−/−) cells. | Other | biomedical | en | 0.999995 |
10085299 | Rat Cx43 cDNA (provided by Dr. Eric Beyer) was subcloned into the PGEM 7Zf (+) vector ( Promega Corp. ) at the EcoRI site. The deletion mutant of Cx43 Δ245 was made by PCR using primers incorporating a stop codon TGA at amino acid 245, before subcloning into the pBluescript IISK (+) vector (Stratagene). The cDNA encoding the COOH-tail of Cx43 244–382 was designed by mutating the codon 5′ to amino acid 245 to an ATG through PCR amplification, followed by subcloning into the pBluescript IISK (+). Mutation at this site also created a consensus Kozak site for translation initiation. The Δ241–280 mutant was made in the PGEM 7Zf (+) vector by PCR using outer universal primers to nucleotides 225–243 and 902–922 of the wild-type sequence, and forward and reverse mutagenic primers. The mutagenic primers were complementary to nucleotides 5′-712 GATCGCGTG 720-3′ and 5′-841 ATGTCTCCTC 850-3′, thus causing the intervening 120 nucleotides to be looped out (making a 40 amino acid deletion). The double point mutants, S255/257A, S279/282A, P253/256A, and P277/280A were also made by PCR using the outer universal primers, and forward and reverse mutagenic primers that mutated both codons. The quadruple serine mutant (S255/257/279/282A) was created by using the mutant S279/282A as the template, and primers that mutate S255 and S257 sites. Tyrosine mutants of Cx43, Y265F, and Y265/247F were constructed in a similar way from a Cx43 template containing an HA tag at the 3′ end. All other mutants were kindly provided by Drs. Steve Taffet and Mario Delmar (State University of New York Health Science Center at Syracuse). The cDNA for pp60 v -src was provided by Dr. Marilyn Resh (Memorial Sloan-Kettering Cancer Center, NY). All cDNAs were linearized with restriction enzymes downstream of the coding region. In vitro transcription was performed using mMESSAGE mMACHINE Kits (Ambion) according to the manufacturer's recommendations. The resultant cRNAs were quantitated after DNase treatment using both OD 260 nm measurement and estimates from Ethidium Bromide stained nondenaturing agarose gels using an RNA ladder ( GIBCO BRL ) as a standard. Adult female Xenopus toads were unilaterally dissected and approximately one-third of the oocytes on that side were removed. The oocytes were treated with 1 mg/ml collagenase ( Sigma Chemical Co. ) to digest most of the follicular cell layers. Oocytes were preinjected with 40 nl of 0.2 μg/μl of an oligonucleotide complementary to Xenopus Cx38 nucleotides 5′-75 GCTTTAGTAATTCCCATCCTGCCATGTTTC 45-3′ . After 72–96 h preincubation, 40 nl of cRNA encoding the connexin construct of interest was injected (0.5–8.0 ng cRNA/oocyte, adjusted to produce comparable coupling levels). The vitelline envelope was than manually stripped before pairing. After 18 h at 18°C, the coupling of the cells was determined by dual cell voltage clamp as detailed previously . After initial recording of conductances, cRNAs for v -src , or v -src (+), the COOH-terminal domain (COOH-tail) of Cx43 (7 ng/oocyte for v -src RNA, 2 ng/oocyte for tail RNA), or an equivalent volume of dH 2 O were injected into the vegetal poles of the paired oocytes. Conductance was recorded again after 6 h incubation at room temperature. In some experiments, it was shown that similar results could be obtained with incubations as short as 3 h. The ratio of conductance post- and pre- src injection was used to determine the effect of v -src on junctional conductance. For more direct comparison with prior studies of Swenson et al. , in one set of experiments v -src cRNA was coinjected with cRNA for either Cx43 or Cx43 Y265F at the same levels as described above. After pairing and 18 h incubation at 18°C, the effects of v -src were expressed as a ratio of the average conductances of oocyte pairs receiving both connexin and v -src cRNAs, and those injected with only connexin cRNA. Comparisons were made within the same batch of oocytes. To assess the probability that the result obtained with each mutant is the same as seen in wild-type or another mutant construct, a t test was performed to determine the P value at a significance level of α = 0.01. Oocytes were injected with RNAs as described above, together with [ 35 S]methionine (2–10 μCi/oocyte; 250 μCi/μl; Nycomed Amersham ), and incubated at room temperature for 6 h. For each experiment, approximately six labeled oocytes were homogenized in 200 μl/oocyte of modified RIPA buffer composed of 0.25% SDS, 50 mM tris(hydroxymethyl)-aminomethane (Tris), pH 7.4, 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 40 mM β-glycerophosphate, 1 mM Na 2 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml pepstatin A, 20 μg/μl leupeptin, and 20 μg/ml aprotinin. The homogenate was brought to 2% Triton X-100 after boiling for 5 min and cleared in a microcentrifuge at 13,000 rpm for 5 min. When nondenaturing immunoprecipitation was performed, oocytes were homogenized in the same buffer, except that 0.1% SDS was used together with 1% NP-40 and 0.5% sodium deoxycholate (final concentration) in the original homogenization buffer. The homogenate was cleared without boiling. 1 μl primary antibody/oocyte (either crude antisera against Cx43 residue 302–319, or monoclonal v -src antibody; Upstate Biotechnology Inc.) was added to the supernatant. After overnight incubation on a rotator at 4°C, preswollen protein A–Sepharose CL-4B beads ( Sigma Chemical Co. ) were added, followed by an additional 1.5 h incubation. The beads were then washed three times in the same RIPA buffer used for oocyte lysis, before solubilization of the immunoprecipitated material by boiling for 10 min in 2× SDS sample buffer (12.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 20% 2-mercaptoethanol, 1 mg/100 ml bromphenol blue) and subsequent separation by SDS-PAGE . The dried gel was analyzed by autoradiography, or was exposed to a PhosphoImaging cassette (model 425E using ImageQuant v.4.2 software; Molecular Dynamics Inc.) for several hours and then bands were quantitated after reading on a PhosphoImager. For alkaline phosphatase treatments, anti-Cx43 immune complex still bound to Sepharose was washed three times with RIPA buffer and twice with phosphatase reaction buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 150 mM NaCl, pH 8.0) supplemented with 0.1% Triton X-100, 0.05% SDS, and 2 mM PMSF; Musil et al., 1990 ). The pellets were then resuspended in 10 μl of phosphatase reaction buffer supplemented with 1% SDS, 1% 2-mercaptoethanol, and 2 mM PMSF. The immunoprecipitated Cx43 was eluted from the beads after heating at 60°C for 3 min followed by dilution with 40 μl of phosphatase reaction buffer, and incubated with 10 U of calf intestinal alkaline phosphatase ( Promega Corp. ) at 37°C for 4 h. Control samples were incubated under identical conditions without alkaline phosphatase. Samples (17 μl) of treated and untreated preparations were then subjected to SDS-PAGE analysis as described above. Oocytes were injected with Cx43 RNA and labeled with [ 35 S]methionine (>1,000 Ci/mmol, 1.7 μCi/oocyte) as above. After 6–7 h of incubation at room temperature, 40 nl of 2.5 mM L -methionine (∼100-fold excess over [ 35 S]methionine) was injected into the vegetal pole. In some experiments, oocytes were allowed to recover from injection for 30 min before addition of cycloheximide to a concentration of 15 μg/ml, previously shown to fully inhibit total protein synthesis in oocytes . Oocytes were incubated at 18°C, and oocyte batches were removed at different time points for immunoprecipitation with anti-Cx43 antisera, analysis by SDS-PAGE, and quantitation of bands as described above. The half-life of Cx43 was found to be slightly longer in the absence of cycloheximide, suggesting that the latter was needed for complete block of synthesis of new labeled proteins. pp60 v -src associated with Cx43 was detected by Western blot after nondenaturing immunoprecipitation using anti-Cx43 antisera. The immunoprecipitate was resolved by SDS-PAGE and then transferred to an Immobilon membrane ( Millipore Corp. ) in transfer buffer (25 mM Tris-base, 192 mM glycine, 15% methanol) at 200 V for 45 min. Membranes were blocked for 1 h in 5% nonfat dry milk in 0.1% Tween-PBS and washed with the same buffer. A 1:500 dilution of anti–v -src mAb was added in 0.1% Tween-PBS buffer and, after 1 h incubation at room temperature, the membrane was washed extensively in the same buffer. Blots were then incubated with a 1:5,000 dilution of sheep anti–mouse secondary antibody conjugated to HRP (ECL kit; Nycomed Amersham ) for 1 h. After further washings, cross-reactive bands were detected using the enhanced chemiluminescence protocol suggested by the manufacturer. NRK cells expressing a temperature-sensitive form of v -src oncogene were cultured in DME with 10% FCS ( GIBCO BRL ) in a humidified 5% CO 2 incubator. LA25-O25 cells are a clone of LA25 cells cotransfected with plasmids containing rat Cx32 cDNA driven by SV40 promoter and hygromycin-B–phosphotransferase by lipofection ( Boehringer Mannheim Corp. ). Clones were selected in DME, 10% FCS, and 400 μg/ml hygromycin B and maintained in the same medium with 300 μg/ml hygromycin B ( Calbiochem , CA). The expression of Cx32 was confirmed by Western blot, immunofluorescence, and dye coupling. All cultures were started at 37°C for 24 h before transfer to the experimental temperatures of 40°C or 33°C for restrictive or permissive growth, respectively. Cells were maintained at either 40°C or 33°C for at least 24 h before coupling was assessed. In some studies, cells were transferred from 40°C to 33°C and dye coupling was measured at different time points thereafter. The MAP kinase kinase (MEK) inhibitor PD98059 (50 μM; Calbiochem-Novabiochem ) in DMSO, or DMSO alone as control, both added to 0.1% (vol/vol) in the culture medium, were used to pretreat cells for 1 h before transferring from restrictive to permissive temperature. Confluent monolayers maintained at either 40°C or 33°C were microinjected pneumatically with a glass micropipette containing 10% lucifer yellow dye (LY) dissolved in 0.33 M lithium chloride using a Zeiss micromanipulator and Eppendorf pneumatic injector. Dye transfer was assessed on a Zeiss phase-contrast microscope (Axiovert 10) via image capture through MetaMorph Imaging System ( Universal Imaging Corp. ) and quantitation by counting number of surrounding cells receiving LY 2 min after microinjection. Although various studies have implicated phosphorylation as the major event mediating the closure of gap junction channels by the v -src oncogene, they have not established a clear model of how this phosphorylation produces the underlying changes required for gating. It is conceivable that such gating could be achieved by initiation of a propagated conformational change, or alternatively, by mediating an interaction between discrete domains of the connexin protein that leads to the occlusion of the channel. To distinguish between models, we examined the responses to v -src of a series of Cx43 truncations and site directed mutants of the COOH-terminal domain. This region had been previously implicated in pH gating , responses to IGF , and phosphorylation by and binding to v -src . All constructs were expressed in Xenopus oocyte pairs that were first microinjected with wild-type or mutant connexin cRNA that had been titrated to produce similar levels of conductance, and in most cases, protein levels . Preinjection of an antisense oligonucleotide to Xenopus Cx38 ∼4 d before the initial cRNA injection was used to effectively eliminate contributions from endogenous connexin. Paired oocytes were allowed to form stable conductance levels (usually after ∼16 h of pairing) before secondary injection of cRNA for pp60 v -src . The effects of this secondary injection on intercellular conductance were assessed after 6 h and expressed as a fractional decrement of the conductance recorded from the same oocyte pair before introduction of v -src cRNA. In some experiments, comparable results were also obtained after incubations as short as 3 h. With this strategy, each oocyte pair serves as its own control, thereby reducing effects of variability between cells. Control injections of H 2 O caused no change in conductance over the time frame of our recordings. To determine if connexin turnover could play a role during the duration of our experimental paradigm, we measured the half-life of Cx43 in Xenopus oocytes as described in Materials and Methods. In oocytes, this proved to be ∼22 h , or over four times that seen in mammalian cells . Initial experiments used a cold methionine chase in concert with cycloheximide treatment to stop synthesis of new protein. Given reports that cycloheximide itself could lead to stabilization of Cx43 , we also performed some experiments in the absence of cycloheximide. This resulted in an even larger estimate of half-life (∼30 h), a result that probably indicates that incorporation of labeled methionine into newly synthesized proteins was not completely blocked by cold chase alone. These measurements demonstrate that our strategy allows us to focus on the gating of established channels during the 3–6-h time period employed, as contributions from protein turnover could be minimal. As reported previously , expression of pp60 v -src in both opposed oocytes caused a dramatic drop (200–500-fold) in conductance formed by wild-type Cx43 . Unilateral injection of v -src RNA decreased the conductance by ∼50-fold (data not shown), a result consistent with the finding that activation of v -src causes a reduction in P o of Cx43 channels (Moreno, A.P., and B.J. Nicholson, manuscript submitted for publication). In contrast, pp60 v -src decreased conductance of Cx32 coupled oocytes by only 37 ± 5% , and cultured cells by ∼30% . A similar effect of v -src was seen in Cx26 expressing oocytes (data not shown). As noted in Swenson et al. , Cx32 contains no tyrosine targets for the v -src kinase, or secondary sites such as consensus serine phosphorylation sites for MAP kinase. Therefore, the decrement in Cx32 and Cx26 conductances mediated by v -src is likely to reflect non-gap junction–specific effects on coupling, e.g., the well-characterized perturbation of adhesion to both substrates and cells by v -src expression. Truncation of Cx43 at residue 245 virtually eliminated the v -src response . This confirmed that target elements of v -src reside between residues 245 and 382 on the COOH-terminal tail of Cx43. Dramatically, the sensitivity of Cx43 Δ245 to v -src was largely restored when cRNA encoding the COOH-terminal tail of Cx43 (244–382) was coinjected with pp60 v -src cRNA. No drop in conductance was shown when the COOH-terminal peptide was coexpressed with Cx43 Δ245 in the absence of v -src (data not shown). Thus, the COOH-tail of Cx43 can function as an independent domain that can occlude the channel upon expression of v -src , reminiscent of the ball and chain mechanism proposed for K + channel inactivation , as well as pH gating and insulin-mediated gating of Cx43 . The COOH-tail domain appears to show specificity for the Cx43 channel, as expression of the Cx43 COOH-terminal peptide failed to induce a drop in conductance in response to v -src in Cx32 expressing oocytes . Insights into the specific molecular mechanism of how the COOH-peptide can mediate v -src induced gating of Cx43 requires a knowledge of which sequences within the COOH-terminal 138 residues that were removed in the Cx43 Δ245 construct are necessary. Initial indications that more than one site might be involved were provided when a less severe truncation of Cx43 at residue 257 (Cx43 Δ257) was tested . This truncation had previously been shown to eliminate the more sensitive pH gating response and insulin induced channel closure of Cx43 . However, it caused only partial reduction of the v -src induced gating of Cx43 compared with that seen with Cx43 Δ245 . As was the case with more severe truncation, the partial loss of v -src gating seen with Cx43 Δ257 was restored by addition of cRNA encoding the missing COOH-terminal fragment (residue 258–382). This shorter COOH-terminal fragment, however, only partially restored the response of more severely truncated Cx43 Δ245 construct , suggesting that the 12 missing residues from 245–257 are important in this process. In a more systematic approach, we examined a series of 20 residue deletions in the COOH-terminal domain of Cx43 . Deletions between 280 and 320 had no effect on v -src induced gating of Cx43 channels . However, two constructs, Δ241–260 and Δ261–280, showed significantly reduced sensitivity to v -src . The first deletion construct, Δ241–260, included the 12-residue site identified from the truncations, although the latter deletion, Δ261–280, showed a more marked effect. A combined deletion from 241 to 280 (Δ241–280) resulted in a greater loss of sensitivity to v -src , yielding a response close to the negative control of Cx32 expressing oocytes . This region includes two proline-rich, putative SH3 binding domains (253–256 and 277–283), along with all of the putative MAP kinase sites , as well as Tyr265 and Tyr247, putative targets of v -src kinase activity . Since the response of Δ241–280 to v -src is not significantly different than that of the Δ245 truncated mutant ( P > 0.01), it is reasonable to propose that relevant residues for v -src mediated gating are located in this region. Furthermore, both of the 20 residue regions identified in the initial deletion series (each containing one of the proline-rich regions and a subset of the serine and tyrosine phosphorylation sites) contribute cooperatively, or at least additively, to v -src gating of Cx43. This was consistent with the comparison of Δ245 and Δ257 truncations of Cx43 described above. Phosphorylation of Cx43 on tyrosines in response to pp60 v -src has been correlated with the loss of junctional coupling in several mammalian cell types . This is consistent with the more direct approach used in Xenopus oocytes, where a mutation of Tyr265 eliminated closure of Cx43 channels by v -src . However, the implication of at least two sites in v -src gating of Cx43 from the above truncation and deletion studies raised questions about ascribing all effects of v -src to a single tyrosine, although a second potential site of v -src phosphorylation (Tyr247) has subsequently been identified . To our surprise, we found that mutants Cx43 Y265F and Cx43 Y247F were inhibited by v -src to the same extent as wild-type Cx43 . Even a double mutant of both tyrosines (Y265/247F) had a similar lack of effect , while Y265F and Y265/247F tyrosine mutants with HA tag at the carboxy end generate functional channels with no detectable difference from wild-type Cx43. These results might seem irreconcilable with those of Swenson et al. . However, in the current experiments we intentionally focused on the acute effects of expressing pp60 v -src after gap junction channels had stably formed, so as to examine predominantly gating events on established channels. This differs from the previous study where v -src and connexins were expressed together, thereby introducing potentially complicating effects on biosynthesis. In a parallel experiment, we attempted to reproduce these conditions by coinjecting cRNAs for v -src and Cx43 or Cx43 Y265F, and recording conductances after 18 h of incubation at 18°C. In this scenario, the Y265F mutant does show a reduced response to v -src (about a fivefold decrease) compared with Cx43 wild-type , although the loss of responsiveness is still less than that reported previously . Together, these results indicate that the known sites of v -src phosphorylation on Cx43 (i.e., Y265 and Y247) are not essential for closure of the channels by this oncogene, although they do appear to play some as yet undefined inhibitory role in biosynthesis of the functional channels. In the absence of evidence relating direct phosphorylation of Cx43 on tyrosines to channel gating by v -src , we turned to an analysis of other sites on Cx43 that could be involved less directly. Both proline rich sequences, 253 PLSP 256 and 277 PLSPMSP 283 , lying within the deleted region we have defined as essential for v -src 's action on Cx43 gap junctions, contain potential SH3 binding domains (PxxP) and putative MAP kinase sites . Two mutagenic strategies were employed in an effort to dissect the relevant functions of these domains. Mutation of prolines in these sites should compromise their function as SH3 binding domains. However, it is also likely to impair their efficiency as targets of MAP kinase, as the prolines form a critical part of the recognition motif for this enzyme. Alternatively, mutation of relevant serines in these sites to alanines should directly eliminate them as targets of MAP kinase. This should have little effect on the proposed role of these regions as SH3 binding elements, based on known characteristics of consensus SH3 binding sites . Thus, one would predict that if the importance of these domains in v -src gating of Cx43 is as a target of MAP kinase, both proline and serine mutations should be equally effective. If SH3 binding is a more relevant property, proline mutants should have a much greater effect on v -src induced gating than serine mutants. Consistent with the former hypothesis, we found that double mutants of either prolines (i.e., P253/256A and P277/280A) or serines (i.e., S255/257A and S279/282A) in either site showed identical refractoriness to inhibition by v -src . A mutant combining serine mutations in both sites (S255/257/279/282A) showed an even greater loss of responsiveness to v -src , compared with mutations within a single site ( P < 0.01 in either case). At an α = 0.01 level of significance, this quadruple serine mutant of Cx43 showed a response to v -src indistinguishable from Cx32 , a connexin containing no consensus sites for v -src or MAP kinase phosphorylation. These results strongly suggest that activation of MAP kinase, or a related kinase with a similar recognition motif, is required for the gating effect of v -src on Cx43. Recent reports have linked the specific binding of v -src to Cx43, by way of both its SH3 and SH2 domains, to the efficient tyrosine phosphorylation of connexin, although the functional consequences for channel function were not assessed . However, the implication of serine kinase sites in the gating of Cx43 by v -src , rather than tyrosine phosphorylation or SH3 binding, leads to the prediction that v -src binding to Cx43 should not have a dominant role in its gating response. This was directly tested by examining the association between pp60 v -src and wild-type and mutant forms of Cx43. To document the same association between Cx43 and pp60 v -src as seen in mammalian cells, Xenopus oocytes were injected with [ 35 S]methionine and Cx43 cRNA, with or without pp60 v -src cRNA . In the absence of pp60 v -src , immunoprecipitation with antibodies to Cx43 (directed to residues 302–319) yield a major band on SDS-PAGE of 43 kD, corresponding to the mobility of nonphosphorylated Cx43 as seen in rat brain , and variable amounts of a lower band of ∼41 kD . This 41-kD band appeared to be a degradation product of the major 43-kD band rather than a phosphorylated variant, as demonstrated by alkaline phosphatase treatment . Upon coexpression of pp60 v -src , a second band of 60 kD was also precipitated by this antibody, but only in the presence of Cx43 . This band comigrates with the pp60 v -src precipitated by an anti- src mAb from oocytes injected only with pp60 v -src cRNA . It was also independently recognized in these Cx43 immunoprecipitates on Western blots probed with v -src antibody (data not shown). Coexpression of v -src and Cx43 also resulted in the appearance of minor bands of slightly slower mobility than the major 43-kD band that have been previously associated with serine phosphorylated forms of Cx43 . This was directly confirmed by alkaline phosphatase treatments that had no effect on the banding pattern of Cx43 in the absence of v -src , but eliminated the slower mobility species seen in the presence of v -src . The increase in intensity of the 41-kD proteolytic product after alkaline phosphatase treatment suggests that this truncated form of Cx43 is also phosphorylated. The 60-kD v -src oncogene was also found to coprecipitate with the COOH-terminal peptide of Cx43 when coexpressed in oocytes , indicating that this domain mediates the interaction of Cx43 and pp60 v -src . As was the case for the full-length connexin, coexpression with pp60 v -src also induced the appearance of a second, slower mobility form of the COOH-terminal peptide that likely corresponds to a phosphorylated form . Each of the mutant constructs tested above was also precipitated from oocytes in the absence (−) or presence (+) of coexpressed pp60 v -src to assess their potential binding capacity . In all mutants, pp60 v -src coprecipitated with Cx43, but in some cases to a much lesser extent. This was quantitated by normalizing the ratio of labeled 60-kD product and labeled mutant Cx43 (in its phosphorylated and partially truncated forms) to that seen with wild-type Cx43 in the same oocyte batch. These results, presented as percentages, are shown in Table I . Only three mutants showed a dramatic loss of v -src binding: both deletions involving the second putative SH3 binding site (Cx43 Δ261–280 and Cx43 Δ241–280); and, to a lesser degree, the double tyrosine mutation Cx43 Y247/265F. Minor reductions in v -src binding were also detected in each of the single tyrosine mutants, and to a lesser degree in the two double proline mutants. No significant reduction in v -src binding was seen in deletions of the first putative SH3 binding domain (i.e., Cx43 Δ241–260) nor in the various point mutations of serines. Consistent with the findings of Kanemitsu et al. , these results suggest a degree of cooperativity between potential binding targets for v -src , including both Tyr265 and Tyr247, and the more COOH-terminal of the putative SH3 binding domains. A comparison of the loss of v -src binding to Cx43 and its functional effect on channel gating (Table I ) reveals a distinct lack of correlation. Some mutants do affect both binding and gating. However, the Cx43 Y265/247F mutant shows markedly reduced binding of pp60 v -src compared with wild-type, but nonetheless closes in response to v -src indistinguishably from wild-type. In contrast, each of the paired site mutants of serines, as well as the Δ241–260 deletion, show significantly reduced gating in response to v -src , but no detectable decrease in v -src binding. This comparison supports the initial prediction, based on a mechanism mainly involving a mitogen-activated or related kinase, that binding of v -src to Cx43 does not play a major role in direct gating of Cx43 channels. We attempted to directly address the role of MAP kinase as an effector of v -src mediated closure of Cx43 channels by inhibition of MAP kinase in Xenopus oocytes, using either antisense oligonucleotides against the ERK2 isotype of MAP kinase, or the MEK inhibitor PD98059. Although inhibitory effects on MAP kinase correlate with reduced ability of v -src to close Cx43 gap junction channels, full inhibition of MAP kinase could not be achieved with either approach in oocytes. Hence, we turned to better characterized mammalian cell lines, specifically NRK cells expressing a temperature-sensitive variant of v -src (LA25 cells), Cx43, and in some cases, exogenously introduced Cx32. Communication through gap junction in these LA25 cells, as measured by dye coupling, is quickly disrupted upon v -src activation . Consistently, we found cell coupling levels decreased as early as 5 min, and dropped dramatically to 5% of original levels within 30 min of switching to the permissive temperature . Treatment of cells with the PD98059 inhibitor of MEK, before and throughout the shift to permissive temperature for v -src activity, allowed the cells to remain coupled . Some reduction in coupling in response to v -src activation (up to ∼55% of original levels) was evident, even in the presence of inhibitor . As with oocyte studies, we employed cells expressing Cx32 (isolated as a stably transfected clone of LA25 cells designated O25 as a control for effects of v -src not specific to connexins. This clone showed only a modest reduction in coupling (up to ∼75% of original levels) in response to v -src activation that proved insensitive to application of the MEK inhibitor. This is comparable to results with Cx32 expressing oocytes, and indicates much of the drop in coupling seen in LA25 cells in the presence of MEK inhibitor is attributable to effects of v -src not specific for gap junctions. Inhibition of intercellular communication through gap junction channels has long been linked to enhanced cell division and growth in both normal and transformed cells. In the case of several growth factors , the induction of transient uncoupling has been linked to MAP kinase mediated phosphorylation of Cx43 . By contrast, in most tumors and transformed cell lines, the mechanism of uncoupling has remained obscure. A notable exception is v -src mediated transformation that is associated with rapid uncoupling of cells correlated with the appearance of tyrosine phosphate on Cx43 . This phosphorylation of Cx43 apparently requires direct association of Cx43 with pp60 v -src . Here we have used the Xenopus oocyte expression system to further investigate the molecular mechanism by which pp60 v -src causes closure of Cx43 channels. Our results implicate a ball and chain model in this gating process, in that an independently expressed COOH-tail peptide restores the sensitivity to v -src of a COOH-terminally truncated form of Cx43. In the originally proposed ball and chain model of K + channel inactivation , gating is mediated by interactions between the channel pore and the gating domain. In the case of Cx43, the COOH-terminal tail serves as the gating particle, with the interaction triggered by v -src expression. To investigate the nature of this triggering, we employed systematic truncation and deletion mutagenesis of the Cx43 COOH-terminal domain. This implicated two regions (241–260 and 261–280), each containing potential sites for tyrosine and serine (e.g., MAP kinase) phosphorylation, as well as SH3 binding motifs. These regions appeared to act cooperatively to fully account for v -src gating of Cx43. While a similar ball and chain mechanism has been proposed to be triggered by reduced cytoplasmic pH , the COOH-terminal sequences that are required are somewhat different from this study , and include residues 374–382, as well as an overlapping domain from 261–300. Thus, it is likely that pH and v -src induced gating utilize different downstream factors requiring distinct structural elements on the COOH-terminal peptide. Recently, an insulin-stimulated decrease in Cx43 mediated coupling was also shown to occur through a ball and chain mechanism . In this case, one of the two domains implicated here (261–280) was found to play a role, although no indication of involvement of residues 241–260 was evident, indicating some differences in this case, too. Whether different receptors for the ball are needed remains to be determined. However, in the case of v -src , this study does demonstrate this receptor is specific for Cx43, since the addition of the Cx43 COOH-tail cRNA together with v -src cRNA did not induce any change of conductance in Cx32 channels. At this point, we can not determine whether the COOH-terminal tail simply occludes the channel, or induces a subsequent conformational change upon interaction with other cytoplasmic domains of Cx43. Using site-directed mutagenesis to further define targets of v -src action within the two identified domains, we were surprised to see no significant change in the response of Cx43 to v -src when Tyr265 was changed to phenylalanine. This residue has been identified in several studies as the likely substrate for v -src on Cx43 . Mutation of a second potential v -src target, Tyr247 , alone, or in combination with Tyr265, also failed to decrease v -src gating of Cx43. The disparity between our data and the results of Swenson et al. , who had shown a loss of v -src induced gating with the same Y265F mutant, may have resulted in part from differences in experimental design. We have focused on the response of preformed gap junction channels by injecting connexin cRNAs into oocytes and allowing formation of stable gap junction conductances before introduction of v -src cRNA. To allow for efficient translation of v -src , we measured its effects on Cx43 coupling after 6 h (although similar results were found as soon as 3 h after src injection). Although we record a >200-fold reduction of coupling, given the short half life of Cx43 in other systems, it is possible turnover of the protein could play a role. However, we have directly measured Cx43 turnover in oocytes and found it to be ∼22 h, presumably a reflection of the lower temperature (∼19°C) of this system, and possibly reflecting the semidormant state of these cells. Thus, turnover of Cx43 contributed negligibly to the reduction in coupling we observed. In contrast, previous studies injected v -src and connexin cRNAs at the same time, and therefore effects of v -src on other phenomena, such as gap-junctional biosynthesis, could have been included during the 24 h incubation employed. Although actions of other kinases have been linked to various stages of Cx43 biosynthesis , activation of temperature-sensitive v -src in mammalian cells appears to have no obvious effect on the distribution of gap junction plaques on the plasma membrane (data not shown). However, this does not preclude more subtle changes that could render the docking interface of connexons nonfunctional. There is already precedent that cytoplasmic domains of connexins can modify extracellular docking events . By recreating the conditions of the earlier study (i.e., coinjection of v -src and connexin cRNAs), we do find a significant reduction in the effect of v -src on coupling mediated by Cx43 Y265F (∼6-fold inhibition) compared with wild-type Cx43 . While this reduced response is less than that reported by Swenson et al. , where Cx43 Y265F showed less than twofold reduction in conductance in response to v -src , such minor differences could arise from variations in Xenopus strains or the pp60 v -src variant. Our results support the contention that direct phosphorylation of Cx43 by pp60 v -src can inhibit coupling, but this appears to affect some earlier point in channel assembly and can not account for the acute uncoupling of cells in response to v -src expression. Such acute gating of Cx43 channels, characterized by a rapid decrease in P o (Moreno, A.P., and B.J. Nicholson, manuscript in preparation), appears to be induced indirectly through MAP kinase. Neither in this nor previous studies, have all possible tyrosine targets in Cx43 been systematically eliminated. However, we have tested all tyrosines that have been identified as substrates of v -src in vivo , in vitro , and that lie within the 241–280 residue domain identified as essential for v -src gating of Cx43. None play a role in acute channel closure. Recent studies indicate that tyrosine phosphorylation of Cx43 requires binding to v -src , through an interaction that is dependent on both Tyr265 and the proline-rich region from amino acids 271–287, that appear to serve as targets of SH2 and SH3 domains of v -src , respectively . We have also demonstrated an association between v -src and the COOH-terminal domain of Cx43 in the Xenopus oocyte system dependent on the same sites. However, the ability of v -src to bind Cx43 did not correlate with its functional effects on Cx43, a comparison that was not made in prior studies. Some mutants that showed markedly reduced pp60 v -src binding were still sensitive to the oncogene (e.g., Cx43 Y265/247F), while others that bound v -src indistinguishably from wild-type Cx43 (such as Cx43 Δ241–260, Cx43 S255/257A and Cx43 S279/282A), had markedly reduced gating responses to v -src . This further reinforces the contention that direct interaction of v -src with Cx43 may modulate coupling and tyrosine phosphorylation of Cx43, but not through channel gating. If direct phosphorylation of Tyr265/247 by v -src or its binding with Cx43, is not critical to the gating of Cx43 channels, then what is the mechanism? One possible alternative is serine phosphorylation of Cx43 by other kinases that are activated by v -src . In several studies examining effects of v -src , Cx43 demonstrated increased levels of phosphoserines in addition to phosphotyrosines . Deletion mutants that showed reduced response to v -src contain documented MAP kinase phosphorylation sites (S255, S279, and S282) embedded in the MAP kinase recognition motif, PXSP. Pairwise point mutations of these serines (S255/257 and S279/282), and the surrounding prolines defining the MAP kinase consensus site (P253/256 and P277/280), support the involvement of this kinase, or one with a closely related target site, in the v -src induced gating of Cx43. It appears phosphorylation at more than one site is required, as a quadruple serine mutant reflected a cumulative effect of the two double serine mutants. Some of these mutants, specifically the prolines, are also likely to have compromised the role of these regions as SH3 binding sites. However, as noted above, the effectiveness of various mutants in eliminating v -src gating of Cx43, and compromising v -src binding to Cx43, are not closely correlated. The most direct case for the requirement for MAP kinase in the gating response of Cx43 to v -src , however, is provided by our studies of acute uncoupling of LA25 cells on activation of v -src . Here, a blocker of MEK (and hence MAP kinase activation) eliminated much of v -src induced uncoupling. As in our oocyte studies, Cx32 expressing cells were used as a control for the effects of v -src not specific to connexins. The cells showed ∼25% reduction in coupling insensitive to application of MEK inhibitor. It is likely this reduction in Cx32 mediated coupling, seen in oocytes and NRK cells, results from the well-documented inhibitory effects of v -src on cell adhesion. This is believed to occur through disruption of the cadherin–β-catenin interaction by a mechanism that does not depend on MAP kinase. Given the established relationship between cadherin expression and efficient gap junction formation , the small but consistent loss of coupling between Cx32 coupled cells and oocytes is not surprising, despite the lack of potential v -src or MAP kinase targets or binding domains on Cx32 itself. All manipulations employed that would be expected to eliminate MAP kinase effects on Cx43 (i.e., Δ245, Δ241– 280, S255/257/279/282A in oocytes, and use of a MEK inhibitor in LA25 cells) served to largely prevent src -induced uncoupling. However, we consistently observed a residual uncoupling effect beyond that seen in Cx32 negative controls. This suggests that, although MAP kinase may be necessary for v -src induced gating of Cx43, other factors may also influence coupling. Of note is a recent report implicating c- src , rather than MAP kinase, in acute loss of gap junction communication in Rat-1 fibroblast cells in response to G-protein receptor agonists such as LPA . Previous studies have not shown direct effects of c- src on cell coupling, but it is possible, in some systems, that c- src may work through effectors different from that activated by v -src . The compilation of results presented suggest that MAP kinase, or a related kinase, is necessary for v -src induced Cx43 gating. This is consistent with established mitogenic pathways of pp60 v -src which associates, and phosphorylates, with the adaptor protein Shc that in turn activates Ras/Raf, leading to activation of MAP kinase. This potentially establishes a common element to the regulation of gap junctions during mitogenesis. EGF and PDGF also acutely suppress gap-junctional communication in Cx43 expressing cells . Although activation of c- src by EGF receptor is central to many of its enhanced mitogenic effects , reduction of Cx43 coupling was correlated with serine, not tyrosine phosphorylation. In this case, too, MAP kinase was the prime suspect . Therefore, we propose that MAP kinase may act as a common downstream effector of uncoupling for both tyrosine kinase growth factor receptors and the v -src oncogene. The study presented here also indicates that this gating is not mediated by a propagated conformational change, but by interactions between discrete domains of Cx43 (i.e., ball and chain mechanism), apparently triggered by a serine phosphorylation event. This potentially represents a common mechanism linking the uncoupling of cells to mitogenesis. | Study | biomedical | en | 0.999999 |
10085300 | Rhodamine-conjugated polyethylene glycols of 3.5 kD (Rh-PEG 3.5 kD) and 10 kD (Rh-PEG 10 kD) were prepared as described . Sources of antibodies and peptides were as follows: mouse anti-β 1 (P4C10) and the peptides GRGDSP and GRGESP were from GIBCO BRL . Mouse anti–human α x (LeuM5) was from Organon-Teknika Inc. Mouse anti–human α M β 2 (MAC-1) was from Upstate Biotechnology Co. Mouse anti–human α 5 (SAM1), rat anti–human α 6 (GoH3), mouse anti–human α 4 (HP2/1), and mouse anti–human β 3 integrin (SZ21) were from Immunotech. Phycoerythrin-conjugated F(ab) 2 anti–mouse IgG was from Jackson ImmunoResearch. Mouse anti-β 3 (PPM6/13) was from Biosource International . Mouse anti-β 3 was from Chemicon International. Alexa 488–conjugated F(ab) 2 anti– mouse IgG was from Molecular Probes. LTB4, fMLP, PMA, thrombin, and Ficoll-Hypaque were from Sigma Chemical Co. Mouse anti–chicken β 1 integrin (CSAT) and mouse monoclonal anti–human β 1 integrin (AiiB2) were generous gifts from Dr. Clayton Buck (University of California, San Francisco, CA). Mouse mAb 15/7, which recognizes an activation epitope on human β 1 integrins , was from Athena Neurosciences. Mouse mAb IB4, which blocks the ligand-binding domains of human β 2 integrins , was a generous gift from Dr. Samuel D. Wright (Merck, Rahway, NJ). PPACK was from Calbiochem-Novabiochem , Matrigel from Becton Dickinson , and collagen I from GIBCO BRL . Purified fibronectin was from Vitex International. Fibrinogen was from American Diagnostica Inc. Fibrinogen uncontaminated by Factor XIII, fibronectin, and vitronectin, a generous gift of Dr. Jeffrey Weitz (MacMaster University, Hamilton, Ontario, Canada), was prepared from fibrinogen obtained from Enzyme Research Labs FIBI. It was first adsorbed with gelatin-agarose to remove fibronectin and then passed over an affinity column to remove Factor XIII. The fibrinogen was precipitated with 25% ammonium sulfate, dialyzed against 150 mM NaCl, 20 mM Tris (pH 7.4), adsorbed with an antibody to human vitronectin linked to Affi-gel, and dialyzed. PAGE analysis showed the resulting fibrinogen to be free of fibronectin or Factor XIII. Western blot analysis revealed no vitronectin (data not shown). Gels, ∼1 mm thick, composed of fibrin, Matrigel, or collagen type IV, were formed in cell culture inserts (pore sizes 3 or 8 μm) from Becton Dickinson as described . Fibrin gels were gently washed with PBS to remove any residual PPACK. Fibrin/fibrinogen-coated surfaces were prepared as described and PMN adhesion was measured by phase-contrast microscopy. Close apposition of PMN to fibrin/fibrinogen-coated surfaces was defined as exclusion of Rh-PEG 10 kD from zones of contact between PMN and fibrin/fibrinogen measured by fluorescence microscopy as described . PMN were prepared as described from fresh heparinized blood from healthy adult donors after informed consent. PMN used in these experiments were >95% pure as determined by Wright-Giemsa staining . 10 6 PMN in 100 μl of PBS supplemented with 5.5 mM glucose and 0.1% human serum albumin (PBSG-HSA) were placed in the upper compartment of each insert and incubated for 0–6 h at 37°C in a humidified atmosphere containing 95% air/5% CO 2 . At the times and concentrations specified, chemoattractants, antibodies, and/or peptides were added to the top and/or bottom compartments in 500 μl of PBSG-HSA. At the end of incubations, chambers were shaken to dislodge PMN from the lower surface of the inserts. The medium in each lower compartment was collected and its content of PMN was determined using a Coulter counter . Unless otherwise indicated, all values reported are the average of six different samples from at least three independent experiments. PMN (10 5 cells/200 μl of PBSG-HSA) were incubated in suspension at 37°C for 30 min in the presence or absence of fMLP (10 −7 M) or LTB4 (10 −7 M), transferred to 96-well polystyrene tissue culture microtiter plates (Corning), incubated for 30 min at 4°C in 200 μl PBSG-HSA containing the indicated primary antibody (2 μg/ml), washed three times with PBSG-HSA at 4°C, further incubated for 30 min at 4°C with either Alexa 488–conjugated or phycoerythrin-conjugated rabbit anti–mouse F(ab′) 2 in 200 μl of PBSG-HSA, washed three times again with PBSG-HSA at 4°C, and resuspended at 4°C in 300 μl PBS containing 2% BSA and 0.3 mg/ml propidium iodide to determine cell viability. The contribution of dead cells (usually <2%) was removed from the final data analysis. The mean fluorescence intensity of 3–5 × 10 3 cells was determined using a Becton Dickinson FACSCalibur ® . PMN chemotax through three-dimensional gels composed of reconstituted basement membrane proteins containing collagen IV, laminin, and fibronectin , or collagen I in response to a gradient of fMLP or LTB4. In contrast, PMN chemotaxis through fibrin gels or plasma clots is dependent upon the specific chemoattractant used. fMLP-stimulated PMN do not migrate through fibrin gels or plasma clots, whereas LTB4-stimulated PMN do . Checkerboard analyses confirmed that PMN migrate through these gels in response to a chemoattractant gradient . Placement of equimolar concentrations of both fMLP and LTB4 into the bottom chambers inhibited PMN from migrating through fibrin gels , confirming that fMLP's effect is dominant over LTB4's effect. Commercial fibrinogen contains small amounts of fibronectin and vitronectin. To test whether matrix components other than fibrin are responsible for inhibiting migration of fMLP-stimulated PMN through fibrin gels and plasma clots, we performed additional experiments using fibrin gels formed from purified fibrinogen that contained no detectable fibronectin, plasminogen, Factor XIII, or vitronectin. PMN stimulated with LTB4, but not with fMLP, migrated through gels formed from purified fibrinogen . Moreover, collagen I gels (60 μg/ insert) each containing 10 μg of purified fibronectin did not affect the migration of either fMLP- or LTB4-stimulated PMN, whereas the addition of fibrinogen to such gels blocked migration of fMLP-stimulated PMN (data not shown). These results are consistent with reports that fibrin(ogen) contains sequences that are ligands for β 1 integrins, and confirm that fibrin is the matrix component that inhibits migration of fMLP-stimulated PMN. To examine the roles of β 1 and β 2 integrins in PMN migration through Matrigel , or fibrin , we added antibodies that block β 1 or β 2 integrins to the upper compartment of Matrigel or fibrin-coated inserts together with PMN and measured the number of PMN that migrated into the lower compartment in response to fMLP or LTB4. As expected, mAb IB4, directed against β 2 integrins , blocked PMN migration through Matrigel or fibrin gels in response to LTB4. Antibody IB4 also blocked fMLP-stimulated PMN migration through Matrigel , and did not alter fMLP's inhibitory effect on PMN chemotaxis through fibrin gels (data not shown). These results are consistent with previous reports that anti– β 2 integrin antibodies block PMN migration through endothelia and through gels formed by a variety of extracellular matrix proteins. In contrast, mAbs AiiB2 and P4C10 , which block the common β chain of β 1 integrins (CD29), had no effect on fMLP- or LTB4-stimulated chemotaxis through Matrigel , or on LTB4-stimulated PMN migration through fibrin gels . However, these same anti–β 1 chain antibodies reversed fMLP's inhibitory effect on PMN chemotaxis through fibrin . Control experiments showed that CSAT , a mAb that binds to chicken but not human β 1 integrins, SZ21, an antibody against β 3 integrins , and PM6/13, another antibody against β 3 integrins , did not alter the inhibitory effect of fMLP on PMN migration through fibrin gels . These antibodies also did not affect migration of LTB4-stimulated PMN through fibrin gels (data not shown). Among the antibodies directed against the α chains of β 1 integrins, only those directed against α 5 chains were effective in reversing fMLP's inhibitory effect on PMN migration through fibrin gels . Neither antibodies against α 4 chains nor antibodies against α 6 chains of β 1 integrins affected migration of fMLP- or LTB4-stimulated PMN through fibrin gels or Matrigel . To confirm that β 1 integrins directly interact with fibrin(ogen), we examined the effects of anti–β 1 integrins on the migration of fMLP-stimulated PMN through gels formed of purified fibrinogen, lacking detectable levels of fibronectin, vitronectin, plasminogen, or Factor XIII. Both antibodies directed against β 1 and α 5 chains of β 1 integrins reversed fMLP's inhibitory effect on chemotaxis through these gels. These results are consistent with reports that fibrin(ogen) contains sequences that are ligands for β 1 integrins. The peptide GRGDSP blocks the interaction of β 1 integrins with RGD ligands on matrix proteins . Like antibodies against β 1 integrins, addition of GRGDSP peptide to the medium allowed fMLP-stimulated PMN to migrate through fibrin gels (Table I ). Control experiments showed that GRGESP peptide, which does not block binding of β 1 integrins to fibronectin or other RGD-containing matrix proteins , did not reverse the inhibitory effect of fMLP on PMN migration through fibrin gels (Table I ). Neither peptide affected the number of PMN that migrated through fibrin in response to LTB4 (Table I ) or through Matrigel in response to fMLP . PMN were incubated in control medium or in medium containing fMLP or LTB4 and allowed to adhere to fibrin-coated 96-well plates. In the absence of chemoattractant <1% PMN adhered to fibrin (data not shown). Over 40% of fMLP-stimulated PMN and ∼50% of LTB4-stimulated PMN adhered to fibrin. mAb IB4, which blocks the ligand-binding sites of three different β 2 integrins , inhibited adhesion of fMLP- or LTB4-stimulated PMN to fibrin by 75–80% . In contrast, mAb AiiB2, which blocks the ligand-binding sites of β 1 integrins , had no significant effect on the number of fMLP-stimulated PMN that adhered to fibrin, and enhanced by ∼25% adhesion of LTB4-stimulated PMN to fibrin . These experiments show that β 2 integrins are the primary PMN surface receptors that mediate adhesion of chemoattractant-stimulated PMN to fibrin. The findings presented above indicate that β 1 integrins, and specifically α 5 β 1 integrins, mediate the qualitatively distinct effects of fMLP and LTB4 on PMN adhesion to, and migration through, fibrin gels. To determine whether fMLP and LTB4 differentially affect the activation of β 1 integrins we used mAb 15/7, which recognizes a conformationally determined epitope on activated β 1 integrins . PMN incubated for 30 min with fMLP exhibited a 10–22-fold increase in binding of mAb 15/7 , compared with unstimulated PMN , whereas PMN incubated for the same length of time with LTB4 showed little change over unstimulated PMN with respect to binding of mAb 15/7. Control experiments showed that surface expression of β 1 integrins was stimulated approximately twofold by LTB4 , and approximately threefold by fMLP , whereas β 2 integrin surface expression was stimulated approximately fivefold by LTB4 and approximately ninefold by fMLP . Other studies showed that the extent of expression of the epitope for antibody 15/7 on β 1 integrins was dependent upon the dose of fMLP used to stimulate the PMN, and that 5 × 10 −6 M fMLP induced maximal expression of this epitope (not shown). In contrast, LTB4 concentrations 10–50-fold higher (i.e., 10 −6 to 5 × 10 −6 M) than those used in the experiments described in Fig. 5 did not increase expression of the 15/7 epitope on β 1 integrins (data not shown). We have used exclusion of Rh-PEG 10 kD from zones of contact between chemoattractant-stimulated PMN and fibrin-coated surfaces as a measure of the closeness of apposition of PMN to the underlying substrate . Previously, we reported an inverse correlation between the formation of zones of close apposition between chemoattractant-stimulated PMN and fibrin gels and the capacity of PMN to migrate through these gels . In the present experiments we used exclusion of Rh-PEG 10 kD to test whether antibodies and peptides that block β 1 integrins, and that facilitate migration of fMLP-stimulated PMN through fibrin gels , affect the closeness of apposition of these cells to fibrin. Antibodies against the β chain of β 1 integrins, or against the α 5 chain of α 5 β 1 integrins (not shown), reduced the percentage of fMLP-stimulated PMN that excluded Rh-PEG 10 kD from zones of contact with fibrin from 80% to 20–30% , and reduced the percentage of LTB4-stimulated PMN that excluded Rh-PEG 10 kD from these contact zones from 20% to <2% . These experiments together with those shown in Fig. 5 show there is a direct correlation between the capacity of fMLP or LTB4 to activate PMN β 1 integrins and the capacity of these chemoattractants to promote close apposition between PMN and fibrin (as measured by exclusion of Rh-PEG 10 kD), and to inhibit PMN migration through fibrin gels . Tumor-promoting phorbol esters, like ligands that bind to PMN and macrophage fibronectin receptors, activate α M β 2 (CD11b/CD18) for phagocytosis of C3bi-coated particles , and promote formation of zones of close apposition between phorbol ester–stimulated PMN and fibrinogen-coated surfaces (Table II ). However, phorbol ester–stimulated PMN do not migrate into fibrin gels, even when treated with antibodies against α 5 β 1 integrins (data not shown). These findings suggested that phorbol esters activate α M β 2 integrins for close apposition to fibrin independently of β 1 integrins. To test this prediction, PMN were incubated with or without antibodies against β 1 integrins, allowed to adhere to fibrin- or fibrinogen-coated surfaces in medium containing PMA, and then were incubated with Rh-PEG 10 kD. 77% of PMA-treated PMN formed zones of close apposition on fibrin even when they had been treated with antibodies against β 1 integrins. In contrast, <15% of the PMA-stimulated PMN that adhered to these surfaces formed zones of close apposition when treated with antibodies against β 2 integrins (Table II ). This experiment shows that when suitably activated, β 2 integrins are capable of mediating close apposition between PMN and fibrin-coated surfaces in the absence of β 1 integrin ligation. The different effects of fMLP and LTB4 on PMN adhesion to and chemotaxis through fibrin gels appear to be a consequence of qualitative differences in the effects of these chemoattractants on the activity of β 1 integrins. That is, fMLP activates β 1 integrins , stimulates PMN to adhere closely to fibrin(ogen) , and inhibits PMN chemotaxis through fibrin gels . In contrast, LTB4 neither activates β 1 integrins nor induces PMN to adhere closely to fibrin(ogen) , and stimulates PMN to migrate through fibrin gels . To our knowledge, this is the first demonstration that signals initiated by two chemically distinct chemoattractants with their respective seven membrane spanning/heterotrimeric G protein–coupled receptors exert different effects on the activation state of a specific β 1 integrin, and regulate PMN migration. As shown in Fig. 2 , fibrin(ogen) is unique among the matrix and plasma proteins tested in arresting the migration of fMLP-stimulated PMN. This is particularly notable in the case of fibronectin, a well-recognized ligand for α 5 β 1 integrins. The failure of fibronectin to induce migration arrest suggests that fibrin(ogen) has heretofore unrecognized properties, independent of its ability to bind α 5 β 1 integrins, that are important in its ability to cause migration arrest. DiMilla et al. and Palecek et al. reported that smooth muscle cells migrate optimally on fibronectin-coated surfaces when their integrins bind to these surfaces at intermediate strengths. Weber et al. reported an inverse correlation between the strength of adhesion of chemokine-stimulated monocytes to surfaces coated with the 120-kD RGD-containing fibronectin fragment and the capacity of these cells to migrate across filters coated with this fibronectin fragment. The findings of Keller et al. and of Wilkinson et al. , and those reported in Fig. 6 , demonstrate an inverse correlation between closeness of apposition of PMN to surfaces coated with proteins that express ligands for PMN receptors and the ability of PMN to migrate on or through matrices containing these proteins. Thus, it seems likely that loose versus close apposition between cells and matrix protein–coated substrates reflects weak versus strong adhesion, respectively, between the cells and the substrate. Antibodies against β 2 integrins reduced adhesion, inhibited close apposition between PMA-stimulated PMN and fibrin (Table II ), and blocked PMN migration through fibrin (data not shown). Antibodies against β 1 integrins had no effect on any of these parameters (Table II and data not shown). These results demonstrate that the interaction of activated β 2 integrins with fibrin is both required and sufficient for PMA-stimulated PMN to form zones of close apposition on fibrin (Table II ), and that PMA bypasses the requirement for engagement of activated β 1 integrins by matrix proteins for PMN to form zones of close apposition on fibrin. Although the signal transduction pathways by which chemoattractants regulate PMN β 1 and β 2 integrins remain to be elucidated, our findings lead us to make three suggestions regarding the organization of these pathways. First, antibodies that activate β 1 integrins do not promote adhesion of unstimulated PMN to fibrin, or inhibit LTB4-stimulated chemotaxis of PMN through fibrin gels (unpublished data). These results suggest that signals initiated by both fMLP receptors and activated β 1 integrins are required to inhibit chemotaxis of fMLP-stimulated PMN through fibrin gels. Second, the finding that fMLP and PMA have similar effects on PMN adhesion to and migration through fibrin gels might suggest that the interaction of activated β 1 integrins of fMLP-stimulated PMN with fibrin activates protein kinase C, and that this is the mechanism by which fMLP signals β 2 integrins to bind closely to fibrin. However, Laudanna et al. reported that calphostin C, a protein kinase C inhibitor, blocks adhesion of PMA-stimulated, but not of fMLP-stimulated, mouse lymphocytes transfected with fMLP receptors, to VCAM-1–coated surfaces. (Adhesion of chemokine-stimulated lymphocytes to VCAM-1 is mediated by activated α 4 β 1 integrins.) Laudanna et al. identified rho as a key participant in fMLP- and IL-8–mediated activation of α 4 β 1 integrins in mouse lymphocytes. This finding suggests to us that rho acts downstream of Gα i in activating β 1 integrins. The report of Caron and Hall that rho participates in coupling CR3 (CD11b/CD18) to the actin cytoskeleton suggests that rho also affects β 2 integrin–mediated functions. Whether PMN LTB4 receptors activate rho is unknown and should be investigated. Third, binding of fMLP to its receptor activates Gα i . The specific Gα activated by LTB4 in PMN has not been reported. Pertussis toxin, which inactivates Gα i , blocks most effects of LTB4 and of fMLP on human PMN. Thus, the finding that fMLP activates β 1 integrins while LTB4 does not suggests that binding of LTB4 to its receptor activates Gα subunits other than, or in addition to, Gα i and that this difference in Gα subunit utilization is responsible for the divergent effects of fMLP and LTB4 on β 1 integrin activation , and on closeness of PMN adhesion to fibrin . Indeed, Arai and Charo have shown differential utilization of Gα subunits after MCP-1 or IL-8 stimulation of MCP-1 or IL-8 receptor transfected HEK293 cells, and Yokomizo et al. have demonstrated that pertussis toxin treatment does not ablate Ca 2+ increases stimulated by LTB4 in LTB4 receptor-bearing CHO cells. Our studies suggest at least three distinct mechanisms by which fMLP could inhibit PMN migration through fibrin gels. First, the combined strengths of adhesion of activated β 1 and β 2 integrins to fibrin could be sufficient to immobilize PMN on fibrin. Our unpublished finding that antibodies that activate β 1 integrins do not inhibit migration of LTB4-stimulated PMN through fibrin gels casts doubt on this combined-strength-of-adhesion hypothesis as an explanation for the inhibitory effect of fMLP on PMN chemotaxis through fibrin. Second is the possibility that binding of fMLP or LTB4 to its cognate receptors directly and differentially activates β 2 integrins for strong or weak adhesion, respectively. According to this hypothesis, fMLP-activated β 1 integrins play no role in inhibiting chemotaxis of fMLP-stimulated PMN through fibrin. However, since activated β 1 integrins mediate outside-in signaling, RGD peptides and antibodies against β 1 integrins reverse fMLP's inhibitory effect on PMN migration through fibrin by stimulating β 1 integrins to signal trans-dominant negative effects on β 2 integrins. Against this hypothesis are the findings that antibodies against the α 6 chains of β 1 integrins , and antibodies that activate β 1 integrins (unpublished data), do not reverse fMLP's inhibitory effect on PMN chemotaxis through fibrin. Third, and we think most likely, is that the capacity of fMLP to promote close adhesion to, and to block migration through, fibrin gels is mediated by a cascade of signals , in which the interaction of activated β 1 integrins with the fibrin matrix causes trans-dominant activation of β 2 integrins. This mechanism is consistent with previous studies showing that interaction of PMN or macrophages with RGD-containing matrix proteins activates α M β 2 integrins for phagocytosis of C3bi-coated particles. As shown in Fig. 7 , we suggest that the interaction of LTB4 or fMLP with their respective PMN receptors generates a “common” signal that activates β 2 integrins for loose adhesion to fibrin . In addition, we propose that fMLP receptors also signal activation of α 5 β 1 integrins . We further suggest that binding of activated α 5 β 1 integrins to fibrin matrices clusters these integrins, thereby generating an outside-in signal that activates β 2 integrins , for close apposition between PMN and fibrin-coated substrates . Close apposition reflects tight adhesion , presumably mediated by the coupling of β 2 integrins to the cytoskeleton. We do not know whether tight adhesion causes, or is merely associated with, cessation of migration. In either case, PMN cease migrating . We propose that antibodies and peptides that block the interaction of activated α 5 β 1 integrins with fibrin inhibit these outside-in signals, thereby blocking trans-dominant activation of β 2 integrins for close apposition to fibrin and allowing PMN to migrate through fibrin. The interaction of LTB4 with its receptor also generates a signal that stimulates β 2 integrins for loose apposition. However, LTB4 does not activate β 1 integrins . Therefore, these β 1 integrins do not bind to the matrix, do not generate outside-in signals, and therefore do not initiate trans-dominant activation of β 2 integrins for close apposition , or cessation of migration. Further work is needed to determine whether cessation of migration is merely a function of strong adhesion between PMN and fibrin or whether it reflects reorganization of the PMN cytoskeleton as observed by Dustin et al. in antigen-sensitized T lymphocytes. They found that these cells become immobilized when they encounter MHC class II molecules containing a peptide antigen recognized by the T lymphocytes' antigen receptors. They identified changes in microtubule organization of these sessile T lymphocytes that distinguish them from their randomly migrating brethren. We suspect that PMN that adhere to fibrin after fMLP stimulation will exhibit similar changes in cytoskeletal organization. Our findings suggest that the availability of many different chemoattractants (e.g., fMLP, LTB4, IL-8, C5a, etc.) serves two complementary functions. First, they provide redundancy, thereby assuring that pathogenic microbes are detected rapidly by the innate immune system. Second, they reflect the need to direct PMN to different tissue sites and to prepare them for interactions with many different types of ligands. Our findings also suggest an alternative to the notion that leukocyte accumulation at a specific anatomic site in vivo requires the presence of a gradient of chemoattractant/ chemokine emanating from that site. While there is no doubt that gradients of chemoattractants/chemokines are formed in vitro , they may be difficult to maintain in vivo in the face of the perturbing effects of muscular contraction and variations in blood and lymph flow. Leukocytes in the vascular system begin to enter specific tissue compartments when they encounter a chemoattractant/chemokine. We suggest that once within this tissue compartment leukocytes migrate randomly in response to a relatively uniform concentration of matrix-bound chemoattractant/chemokine. When in the course of this random walk they encounter extracellular matrix proteins or cells that express ligands for a specific activated β 1 integrin, they adhere strongly and become sessile. By regulating activation of specific receptors and adhesive strengths, concentrations of chemoattractants/chemokines well below those required to saturate or desensitize chemoattractant/chemokine receptors can mediate a stochastic process by which leukocytes accumulate at specific anatomic sites and form highly ordered structures (e.g., granulomas, germinal centers). According to this model, leukocytes accumulate at specific anatomic sites by a process that is similar in principle to the accumulation of flies on fly paper. Foxman et al. showed that multiple chemoattractants/chemokines can work in combination to elicit migration patterns that cannot be achieved by a single chemoattractant/chemokine. The mechanisms we and they have described are complementary. These mechanisms are likely to be of special importance within tissue compartments where overlapping fields of chemoattractants/chemokines/cytokines surely occur, and where cells migrate in stepwise fashion from one anatomic site to another , PMN accumulation at foci of bacterial infection, or of immune-complex deposition . The essential point of the findings reported here is that by endowing leukocytes, and probably all migrating cells, with a modest number of receptors for different chemoattractants, chemokines, and cytokines, nature has made optimal use of instructive and selective mechanisms to achieve a level of organizational specificity that would otherwise require substantially more genetic information. | Study | biomedical | en | 0.999997 |
10085301 | Stock cultures of clone C of β-intercalated cells established from rabbit kidney cortex were maintained as described . The cells were trypsinized and seeded on polycarbonate filters (pore size, 0.4 μm; Costar Corp.) at a density of 2 × 10 4 cells/cm 2 (low density), or 10 6 cells/cm 2 (high density), and transferred to 40°C to inactivate the T antigen. At temperatures between 37° and 40°C, no positive staining of the T antigen was observed by immunofluorescence or immunoblot analysis. High or low density cells on polycarbonate filters were pulse-labeled with 100 μCi/ml of [ 35 S]methionine added to both apical and basal media ( 35 S-protein labeling mix; DuPont-NEN ) for 5 min at 40°C at various time intervals. Cells were lysed in buffer A (1% SDS, 1 mM EDTA, 1% Triton X-100, 10 mM Tris-HCl, pH 8.0) and boiled for 3 min. Insoluble materials were removed by a brief centrifugation (14,000 g for 5 min at room temperature) and the protein concentration of the supernatants was determined by the Bradford reagent (Bio-Rad Laboratories). An equal amount of protein was taken from each sample, diluted 10-fold with 10 mM Tris-HCl, pH 8.0, and used for immunoprecipitation. Clone C cells seeded at high or low density were cultured for 5 d and labeled with 35 S-protein labeling mix added to both apical and basal media for 12 h. Apical and basolateral media were collected separately and centrifuged at 5,000 g for 5 min at 4°C. The supernatants were mixed with 1/10 vol of buffer A and analyzed by immunoprecipitation. Samples from the pulse labeling experiments and secretion studies were incubated with 1:500 dilution of guinea pig anti-hensin antiserum at 4°C for 1 h. Immunoprecipitates were collected by mixing the samples with protein A–Sepharose CL-4B ( Pharmacia Biotech, Inc. ) at 4°C for 1 h. The beads were washed with a buffer containing 0.1% SDS, 0.1 mM EDTA, 0.1% Triton X-100, and 10 mM Tris-HCl, pH 8.0. Immunoprecipitated protein samples were extracted from the beads by boiling them in SDS-PAGE sample buffer for 3 min. Thus, samples extracted were subjected to SDS gel electrophoresis in 7.5% gel. Gels were fixed with 10% acetic acid, 10% methanol, soaked in solution (Amplify; Amersham Pharmacia Biotech ), dried, and exposed to X-ray film (Kodax X-OMAT) at −80°C. Films were developed and analyzed using a densitometer (model 300A; Molecular Dynamics, Inc.). Intermediate filament fraction was prepared from confluent monolayer cultures of both low and high density cells with a modified procedure of previously published protocols . In brief, monolayers were lysed with a solution containing 10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 5 mM EDTA, 15 mM β-mercaptoethanol, 1 mM PMSF, and 1% Triton X-100 for 3–5 min at 4°C. The lysis buffer was aspirated and the monolayer was extracted with a high salt buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1.5 M KCl, 5 mM EDTA, 0.5% (wt/vol) Triton X-100, 15 mM β-mercaptoethanol, and 1 mM PMSF, at 4°C for 1 h. The extract was centrifuged for 30 min at 3,000 g (4°C) and the pellet was washed with buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, and 5 mM EDTA). The final pellet was dissolved in SDS-PAGE buffer, the sample was electrophoresed in a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with anticytokeratin19 antibody . These samples were prepared from an equal number of cells. The immortalized intercalated cells (clone C) were plated at high or low density and cultured for 1–2 wk at 40°C on Transwell filters, depending on the experiment. The following procedures were performed at room temperature: cells were fixed in 4% paraformaldehyde for 10 min, blocked, and permeabilized in a solution of 3% BSA and 0.075% saponin in PBS, pH 7.4, for 1 h. The Transwell filters were incubated in primary antibodies diluted 1:100 in the PBS/BSA/saponin solution for 1–2 h. The following primary antibodies were used: mouse mAb to E-cadherin , fodrin , cytokeratin19 , villin and rat anti-ZO1 antibody (all from Chemicon International, Inc.) and anti–β-tubulin antibody ( Boehringer Mannheim GmbH ). Fluorescein- or rhodamine-labeled goat anti–mouse IgG and rhodamine-labeled donkey anti–rat IgG (Jackson ImmunoResearch Laboratories) were used as secondary antibodies. For F-actin staining, the filters were incubated with 300–600 U of rhodamine-phalloidin (R-415) (Molecular Probes, Inc.). Stained monolayers were mounted on glass slides with 90% glycerol in PBS with 0.1% phenylene diamine and viewed by an Axiovert 100 laser-scanning confocal microscope (model LSM 410; Carl Zeiss ). Excitation was accomplished with an argon-krypton laser producing lines at 488, 568, or 647 nm. Rhodamine- and Cy3-labeled samples were viewed with the 568-nm channel (red), whereas the fluorescein–Hoechst 33342-labeled samples were viewed with the 488-nm channel (green). In the case of triple labeling with Hoechst/hensin and collagen type IV, the Cy5-conjugated collagen type IV antigens were visualized with the deep red 645-nm channel. The images were collected at 1 μm thickness optical sections and analyzed by the Zeiss LSM-PC software. The final images were processed with Adobe Photoshop software. Guinea pig anti–hensin antibodies were obtained as described earlier . A fusion protein containing scavenger receptor cysteine rich (SRCR) domains 5 and 6 of hensin was used to generate these antibodies. The immortalized intercalated cells (clone C) were plated at high or low density and cultured for 1–2 wk at 40°C on Transwell filters depending on the experiment. In the studies aimed at determining the extracellular accessibility of hensin, the filters were first incubated with 10–20 μg/ml of Hoechst 33342 dye (Molecular Probes, Inc.). After extensive washing with PBS, cells were incubated in 1:50 dilution of guinea pig anti-hensin antibody solution in PBS alone, or with 1:100 dilution of the mouse anti–collagen type IV mAb overnight at 4°C. The filters were washed with PBS and incubated with rhodamine–Cy3 conjugated anti–guinea pig IgG (Jackson ImmunoResearch Laboratories, Inc.) alone, or with Cy5 conjugated anti–mouse IgG (for collagen IV triple label experiment) for 1 h at room temperature. The filters were washed, fixed for 30 s–1 min with ice-cold methanol, washed extensively with PBS, and mounted and analyzed with confocal microscopy as described before. Control experiments were performed to test whether prolonged incubation at 4°C resulted in cellular permeabilization. When the guinea pig anti-hensin antibody and anti–β-tubulin mAb were incubated together under the same conditions, we found no specific staining for tubulin, whereby indicating that the cells remained impermeable to these molecules. For studies involving the determination of cellular localization of hensin, the confluent monolayers of high or low density clone C cells were first incubated in Hoechst 33342 at 10–20 μg/ml for 1 h at room temperature, washed with PBS, and fixed with ice-cold methanol for 1 min. After extensive washing with PBS, cells were permeabilized by incubating with a solution of PBS/BSA/saponin (as described above) and incubated further with guinea pig anti-hensin antibody, followed by rhodamine–Cy3 conjugated anti–guinea pig IgG. Prostate glands and intestinal tissues obtained from male New Zealand white rabbits (Hare Marland) were embedded and frozen in tissue-tek OCT compound (Miles Laboratories) until cut into 5-μm cryostat sections. The sections were fixed in ice-cold methanol for 8 min, washed extensively with PBS, and blocked with 10% FCS and 1% donkey serum (Jackson ImmunoResearch Laboratories, Inc.) in PBS for 30 min. The sections were incubated with guinea pig anti–hensin antibody , diluted 1:100 in PBS at room temperature for 2 h and incubated with rhodamine-conjugated donkey anti–mouse IgG. The prostate sections were further incubated with FITC-conjugated monoclonal anticytokeratin7 diluted 1:25 in PBS. Finally, sections were extensively washed and mounted for analysis under a confocal microscope. All analyses of cell size and height were performed with Microsoft Windows ® based version of the Zeiss 410 laser scanning microscope software ( Carl Zeiss GmbH ). The stepper motor and the image analysis software use the internal calibration method for this microscope. All 1-μm sections of a particular field from a sample of phalloidin-stained low or high density cells were projected on the screen together with maximum overlap and all other default settings of the Zeiss LSM-PC software. For size measurements, individual cell boundaries were delineated using a mouse-driven marker (via the Mark Area subfunction of the Area Measure function of this software) and the area obtained in μm 2 is recorded. The area of 10–15 individual cells of each sample were recorded and were analyzed statistically with the program Corel Quattro Pro. For measuring the height of the cells, the xz-section of the sequence of images from each specific sample is used. The two “posit” (position marker) cursors are placed on the same x-coordinate of the xz-section. However, one “posit” cursor is placed on the y-coordinate, corresponding to the first observed apical stain above the nucleus, and the other is placed on the last observed basal stain below the nucleus. The distance measurement function of the Zeiss LSM-PC software is invoked to measure the height. Again, 10–15 individual measurements were recorded for each sample and were analyzed statistically using Corel Quattro Pro. For all these measurements, the phalloidin- or phallodin–Hoechst 33342-stained samples were used to ensure the uniformity of the measurements. Filters were washed in PBS and fixed with 2.5% glutaraldehyde in 100 mM phosphate buffer. The filters were postfixed for 1 h in 1% osmium tetroxide/1.5% potassium ferricyanide and dehydrated through a graded ethanol series: 50, 70, 85, 95, 100, 100, 100% for 10 min at each concentration. Afterwards, cells underwent critical point drying using CO 2 in an Omar SPC-1500 critical point dryer. The filters were cut from their holders and mounted on copper specimen supports, sputter-coated with ∼11 nm of Au-Pd in a sputtering unit , and viewed at 20 kV in an electron microscope (JEOL 100 CX-II; JEOL USA, Inc.) equipped with an ASID scanning unit and images were recorded on Polaroid Type 55 positive/negative film. Guinea pig anti–hensin antisera were diluted 1:100 in the culture medium. Anti-laminin B2 mAb obtained as hybridoma supernatant D18 from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA) was diluted in the culture medium to a final concentration of 30 μg/ml and mouse anti–human collagen type IV mAb was diluted to a final concentration of 60 μg/ml in the culture medium. Confluent monolayers of clone C cells grown at 32°C were trypsinized and centrifuged. The cell pellet was resuspended in the appropriate antibody-containing culture medium and plated at high density (10 6 cells/cm 2 ). Antibody-containing culture medium was replaced daily for 5 d and the cells were processed for immunocytochemistry as described above. Hensin containing ECM-coated filters were obtained through the following procedure: (a) High density cells cultured on Transwell filters for 1 wk were extracted with 1% Triton X-100, 1 mM calcium chloride for 1 h at 4°C on a rotary shaker. (b) Cell extracts were removed and filters were scraped in this solution with a cell scraper to remove loosely attached materials. (c) Filters were washed thoroughly with the same solution for another hour at 4°C followed by three washes with the culture medium. (d) The filters were left overnight in the culture medium on a 4°C-rotary shaker and clone cells were seeded on such filters and cultured as usual. Murine laminin-coated Transwell filters, human fibronectin coated Transwell filters, and Matrigel basement membrane matrix- (from Engelbreth-Holm-Swarm Mouse Sarcoma) coated cell culture inserts were obtained from Becton Dickinson Labware . The laminin- and fibronectin-coated filters were rehydrated by incubation with culture medium for 30 min at room temperature before seeding the cells. In the case of Matrigel-coated filters, the filters were thawed overnight on a level surface at 4°C and the Matrigel solution from one Transwell filter was spread on two Transwell filters before rehydration and seeding. This was necessary as the thick gelatinous layer of matrigel present on the precoated filters prevented the immunohistochemical analysis of the cells cultured on them. Initial studies showed that high density ECM contained a new protein (hensin) that induced the reversal of polarity, but the low density matrix did not. We generated antibodies to a hensin fusion protein containing SRCR domains 6 and 7 that were used for immunoprecipitation and immunocytochemistry. Fig. 1 shows the results of immunocytochemistry using confocal microscopy. Low density monolayers incubated with the anti-hensin antibodies, in the absence of detergents, showed no hensin staining. Therefore, the absence of hensin in the extracellular space of these cells demonstrates and confirms our previous biochemical results . Low density cells like other epithelia, secrete collagen IV into their ECM, was found to be accessible to anti–collagen IV antibodies in the absence of detergents . To examine whether hensin was present in the extracellular space of high density cells, we exposed the cells to anti-hensin antibody in the absence of detergents. Hensin was found to be accessible to the antibody and its distribution was similar to that of collagen IV . Note that both hensin and collagen IV were deposited on the filters as shown by the colocalization in the xy confocal planes . These results demonstrate that hensin localizes to the basal and lateral regions of the extracellular space of high, but not low density cells and overlaps in distribution, at least partially, with a bona fide ECM protein. Surprisingly, when the cells were permeabilized by saponin, we found that even low density cells contained a large number of hensin-containing intracellular vesicles located diffusely throughout the cell, including the apical half. On the other hand, the high density cells contained few intracellular vesicles staining for hensin. This difference in distribution started at the earliest time examined (3 h of plating) and reached the final distribution within a few days. To examine the initial rate of hensin synthesis, cells were plated at high or low density on nucleopore filters and labeled with a short pulse of [ 35 S]methionine at various times after seeding. We found that low density cells, reproducibly, had higher levels of synthesis . To examine the secretion of hensin, the cells were labeled for 12 h with [ 35 S]methionine and the apical and basolateral media were collected and immunoprecipitated. Both phenotypes secreted hensin in a polarized manner to the basolateral medium . The induction of apical endocytosis is the most dramatic and reproducible effect in the conversion of low density phenotype to that of high density. Apical endocytosis, or its lack, clearly reflects large alterations in the apical cytoskeleton. We examined components of this cytoskeleton and found dramatic differences as a function of seeding density . F-actin, in low density cells, was located underneath the basal and lateral membranes but there was only a faint, if any, staining under the apical membranes. High density cells had dense subapical actin network (in addition to the basolateral network). There were more stress fibers in the basal region of low density phenotype, perhaps suggesting that these cells might be less well-differentiated than the high density cells . Remarkably, cytokeratins were also quite different. Cytokeratin19, recently implicated in the organization of the terminal web of intestinal epithelial cells , was largely absent from low density cells but was abundant in high density cells . More significantly, cytokeratin19 was located mostly in the subapical region of the cells. These studies suggest that the terminal web, an actin–cytokeratin mesh critical for the formation of microvilli, was substantially different in the two phenotypes. When the microfilament fraction of low density cells was extracted and subjected to immunoblot analysis, no cytokeratin19 was found, whereas that of high density cells was quite enriched in this protein. This result suggests that high density cells can synthesize this protein . Next, we examined the distribution of villin, the most critical actin-binding protein for microvillar structure , and found that its expression in low density cells was barely detectable; when seen it was expressed in a faint cytoplasmic pattern. In high density cells, villin was highly expressed and located entirely in the apical region of the cell in a manner suggestive of incorporation in microvilli . These studies are reminiscent of the development of the apical cytoskeleton in the intestine; the epithelial cells of the crypt (including stem cells) have essentially no villin or subapical cytokeratin but as they migrate up the villus to become terminally differentiated, they start expressing villin and cytokeratins and develop a thick brush border . This complex pattern of new gene expression suggest activation of a differentiation pathway. Although the above observations might suggest that low density cells are undifferentiated, in fact, these cells are bona fide epithelial cells. They show polarized distributions of proteins , lipids , and polarized secretion of hensin . They are also capable of steady state transepithelial transport of HCO 3 − , which would not be possible without polarized distribution of transport proteins and sufficiently impermeant intercellular junctions . Further, low density cells have all of the following: well-formed tight junctions ; adherent junctions ; a polarized cytoskeleton, where F-actin was present in lateral and basal surfaces ; and fodrin in the lateral membranes . There was also no significant difference in the distribution of paxillin and focal adhesion kinase (FAK; not shown) in the two phenotypes. Intercalated cells in situ do not exhibit brush borders, but scanning electron microscopy showed that their apical cell membranes have impressive specializations: β-intercalated cells have apical microvilli whereas α-intercalated cells have apical folds, termed microplicae . The latter are broad apical ridges that are readily observed in scanning electron micrographs of mammalian kidney and their homologous epithelia in amphibia and reptiles. We performed scanning electron microscopy on low and high density cells and found that low density cells had few apical microvilli scattered over the apical surface that did not change with length of time in culture. Fig. 7 shows low density cells after 1 wk in culture. After 1 wk in culture at high density, the number of well-formed microvilli was much higher and they appeared to be taller and thicker than those in low density cells. In addition, a substantial fraction of the cells had clearly established microplicae . After 2 wk in culture, all high density cells had microplicae of different dimensions and very large microvilli . These results demonstrate that the transition of low to high density phenotypes represents a fundamental change in the properties of the apical cytoskeleton and by analogy with other epithelia, we suggest that it represents a terminal differentiation phenomenon. Differentiation of epithelial cells is associated with changes in cell shape. Indeed, it has been suggested that a change in shape itself might induce terminal differentiation . Protoepithelial cells are flat with a large apical surface area, whereas terminally differentiated epithelia are taller and more tightly packed. This process of columnarization is frequently seen during development . Examination of Figs. 3 , 4 , 6 , and 7 , shows that low density cells are thin and have a large cross-sectional area whereas high density cells are columnar, tall with smaller cross-sectional area. Quantitative analysis of the confocal images is shown in Table I . On average, low density cells have a cross-sectional area of 841 ± 83 μm 2 whereas that of high density cells was 185 ± 12 μm 2 . The height of low density cells was 4.75 ± 0.2 μm, but that of high density cells was 8.73 ± 0.2 μm (mean ± SEM, P < 0.01, Table I ). To examine whether hensin mediates columnarization and cytoskeletal changes, we seeded cells at low density on filters that were coated with a high density matrix. These conditioned filters were prepared by seeding cells at high density for 1 wk, solubilizing with Triton X-100, and discarding remnants. Fresh cells were seeded at low density. Within 1 wk of plating, these low density cells were instructed by the high density matrix to become columnar epithelial cells resembling high density cells. The cell height was 9.06 μm ± 0.2 (Table I ) and the cross-sectional area became 233 μm 2 ± 22. High density matrix was also able to change the actin, villin, and cytokeratin19 distribution in these low density cells so that it resembled that of high density cells . Cells plated at low density on filters pretreated with pure ECM proteins, such as fibronectin and laminin, failed to induce the appearance of subapical actin or change their shape . When the cells were plated on matrigel, a complex mixture of many extracellular proteins (produced by the epithelial EHS murine tumor cell line), there was no columnarization or the appearance of subapical actin . Hence, the conversion of low density phenotype to a high density one, was not merely due to the presence of the known ECM proteins. Hensin was purified using an assay that reflects reorganization of the apical cytoplasm, i.e., apical endocytosis . Further, in high density cells, antibodies to hensin inhibited the development of apical endocytosis while preimmune sera were ineffective . In similar experiments, we seeded cells at high density in the presence of anti-hensin antibodies and examined for the development of subapical actin cytoskeleton. Fig. 9 shows that very little actin localized to the apical cytoplasm and the basal surface of these high density cells now contained many stress fibers similar to the basal surface of low density cells. The anti-hensin antibodies also reversed the change in cell shape that was induced by high density plating. When cells were seeded at high density in the presence of anti-hensin antibodies, their height collapsed to that of low density cells, decreasing to 4.6 ± 0.2 μm and the cross-sectional area increased to 385 ± 23 μm 2 (Table I , n = 11, P < 0.01). Treatment of high density cells with nonimmune guinea pig serum (at the same concentration), or with antibodies to laminin or collagen IV did not prevent the appearance of the subapical actin network, nor did they inhibit the columnarization . These results demonstrate that when intercalated cells are seeded at high density, it is hensin that mediates the reorganization of the actin cytoskeleton and columnarization rather than other ECM proteins. One of the best described terminal differentiation events is the intestinal stem cell ascends to the villus tip to become the terminally differentiated absorptive enterocyte . Because the highest level of expression of hensin is in the intestine , we examined the distribution of hensin in intestinal epithelia. Remarkably, hensin was present in the crypt, largely in a diffuse intracellular pattern that was mostly in the apical half of the cell with no staining of the ECM, similar to the staining pattern of permeabilized low density cells shown in Fig. 1 A. In the terminally differentiated villus cells, hensin was present exclusively in a basolateral pattern likely to represent extracellular localization, an identical distribution to that in permeabilized high density cells. The epithelium of the prostate gland is another tissue that undergoes terminal differentiation in situ. Basal cells are sparsely located in the epithelium and they give rise to the luminal cells, which are terminally differentiated epithelial cells. Basal cells of the rodent prostate gland express cytokeratin7, whereas the luminal cells do not . As can be seen in Fig. 10 B, the rabbit basal cells also express cytokeratin7 (in green); these cells have a large number of vesicles that contain hensin (in red). On the other hand, in the luminal cells, hensin surrounds the cells in a pattern entirely similar to that of high density cells. There is increasing evidence that a number of polarized membrane proteins are targeted in a cell-type specific manner . For instance, the human LDL receptor when expressed as a transgene in mice is located in the apical membrane of kidney epithelia but is basolateral in the intestine and liver . The Na, K ATPase is basolateral in many epithelia, but is apical in the retinal pigment epithelium and choroid plexus . Most GPI-linked proteins are apical but they are basolateral in a thyroid cell line . In the intercalated cell, we demonstrated that the same cell is capable of retargeting at least two proteins, the anion exchanger kAE1 and the H + ATPase to opposite membrane domains, but two other proteins, a basolateral glucose transporter and an apical lectin-binding protein did not change their polarized distribution . These studies demonstrate that there does not appear to be a single class of mechanisms responsible for targeting polarized proteins in all cells. Because the protein that exhibits plastic polarity (e.g., Na, K ATPase or kAE1) is well polarized regardless of whether it is located in the apical or basolateral domain, one can conclude that each protein contains at least two potential targeting signals that are recognized differently by the cell machinery. Another version of this statement is that there may be a hierarchy of signals and that each cell reads these signals depending on its complement of targeting pathways. But remarkably, the cellular machinery that decodes these signals is also cell type specific. For instance, syntaxin 3, a t-SNARE critically involved in delivery of protein to the apical membrane of many epithelia , is present in the basolateral membrane of the intercalated cell . Furthermore, the pathway that vesicles take is also different: the apical targeting of dipeptidyl peptidaseIV is direct in some cells (MDCK), indirect in others , or exhibit both pathways in still others . Recent observations showed that apical epithelial proteins when transfected into nonpolarized cells (fibroblasts) are localized in vesicles or membrane domains that are different from those that contain transfected basolateral proteins . In summary, these studies demonstrate that there are multiple targeting pathways in many, even all cells. To generate a tissue-specific phenotype would probably require local factors that would provide additional information to the basic cellular targeting machinery or might even induce new pathways. To begin to analyze these local factors, it is worth recalling that some of these plastic targeting events occur during development. The Na, K ATPase is found to be localized in the apical membrane in some nephrons during embryonic life and the probability of its basolateral location increases as the kidney matures . A membrane protein in the retinal pigment epithelium changes its polarized distribution during postnatal development . Perhaps the signal for this reversal of polarity comes from a developmentally regulated extracellular instructive molecule. In the retinal pigment epithelium, the N-CAM molecule is apical in situ, but when the epithelium is dissociated from the neural retina and cultured in vitro N-CAM moves to the basolateral surface . Many developmental phenomena are mediated by factors that bind tightly to ECM proteins including: TGFβ family members, wnt's , and fibroblast growth factors. Further, ECM proteins that bind to these critical factors are themselves part of the developmental pathway and serve not only to generate gradients but also as reservoirs, activators, or inhibitors of these factors before they bind to their plasma membrane receptors. Our recent studies had demonstrated that one such protein, hensin, when deposited in the ECM of intercalated cells is sufficient to reverse the polarized distribution of the kidney form of band 3 (kAE1), and the proton ATPase . Hence, one hypothesis to emerge from this analysis is that tissue-specific proteins deposited in the ECM during development might instruct differentiating epithelia to target polarized proteins to one or another domain of the cell. If these ECM proteins are not present in that tissue, then the vesicle targeting pathway will come under the influence of other less dominant factors. To exhibit reversal of polarity, according to this formulation, the protein must contain multiple targeting sequences. These speculations were solidified by the surprising finding of the present paper that hensin appeared to retarget several proteins and induce terminal differentiation in the intercalated cells. Both forms of the intercalated cells are stable when the two monolayers are confluent; all their cells are forming a true epithelial sheet, yet they exhibit different properties. Therefore, hensin must be acting as a binary switch to trigger the conversion of one phenotype to another, a process that is reminiscent of the induction of a differentiation program during development. Epithelial cells of the kidney (and many other organs) begin as mesenchymal cells that are induced to form the elementary epithelial phenotype, proto-epithelial cell. However, these cells only acquire a fully differentiated phenotype later in development. Terminally differentiated epithelia have several characteristics: (a) They contain a rich apical cytoskeletal network, the terminal web, which is composed of an actin mesh and specific cytokeratins. (b) Their apical membrane contains extensive specializations such as cilia, flagella, or brush-border microvilli. (c) They frequently contain vesicles or granules located in the apical half of the cell that are capable of fusion with the apical membrane in a manner regulated by cell calcium or other second messengers. (d) Their nuclei are present in the basal half of the cell. (e) They also assume a recognizable shape in simple epithelia, being columnar or cuboidal. Remarkably, all of these characteristics were induced by hensin. The cells developed a terminal web of actin and cytokeratin19, their apical membranes formed an exuberant microvillar structure, and their shape became columnar. They even developed vesicles capable of regulated exocytosis. We had previously shown that the H + ATPase in the α-intercalated cell is packaged in vesicles that fuses with the apical membrane in response to an increase in the ambient pCO 2 acting to increase cell calcium . We suggest that hensin is the first protein in a new pathway for differentiation in these cell types. It had been known for some time that the ECM is critical for differentiation of epithelial cells. In fact, recent studies showed that fetal intestinal epithelia can start to express terminally differentiated proteins such as villin and cytokeratin when cultured on matrigel, a complex ECM prepared from cancer cells . Hensin is expressed in a large number of epithelial tissues and at a high level in the intestine. Its distribution in intestinal crypt cells is similar to that of low density intercalated cells, whereas in villus cell it was similar to the terminally differentiated intercalated cell. We are presently investigating whether hensin is also involved in the differentiation of intestinal epithelia. Hensin was accessible to its antibodies even when the high density cells were not permeabilized, and its distribution colocalized with collagen IV. How does the high density plating cause precipitation in the ECM? The localization of fibronectin fibrils in the ECM suggests a possible mechanism. Soluble fibronectin monomers cannot form fibrils unless their receptor, the integrin α 6 β 1 is activated . Once activated the receptor's affinity for fibronectin increases, allowing it to bind fibronectin and to form fibrils . Perhaps high density seeding induces a change in the affinity of the hensin receptor that refolds hensin into an insoluble form. Alternatively, high density cells might secrete another protein that will cause precipitation of hensin in the extracellular space. We are presently investigating these two possibilities. The role of the ECM in controlling cell shape is well-established in many cell types including epithelia . Many studies have identified a pathway mediated by binding of integrins to components of the ECM that leads to activation of a signaling pathway mediated largely by activation of FAK. This protein is critical for organization of the actin cytoskeleton and leads to formation of a complex of actin binding proteins such as paxillin, ezrin, and moesin. Cell shape, motility, and ruffle formation are thought to be direct consequences of these events. Most of this information has been obtained in fibroblasts or in blood cells and the role of focal adhesions in epithelia is not as well studied. We found no change in the distribution of paxillin and FAK during the transition from low density to high density phenotypes. However, it was clear that there were large changes in other cytoskeletal components. A dramatic reorganization of the apical cytoplasm occurred that was characterized by all of the following: induction of vigorous apical endocytosis; development of exuberant microvillar formation, the new subapical localization of actin, and the possible induction or at least increased concentration of villin and cytokeratin19 in the subapical cytoplasm. The basal actin cytoskeleton was also different in the two phenotypes. Low density cells had many “stress fibers” in the basal region similar to what is seen in nonepithelial cells whereas the actin network in high density cells appeared to be more diffuse. How a basolateral extracellular molecule induces these changes in cell shape and the changes in apical cytoplasm is unknown at present. Recent studies have suggested that mechanical stress, such as that following binding of ECM proteins to their receptors have profound effects on cell functions, including the induction of complex programs of differentiation . The cellular mechanisms by which hensin induces these events awaits the identification of its signal transduction pathway and preliminary evidence suggests that a basolateral integral membrane glycoprotein acquires tyrosine phosphorylation within a short time after seeding these cells at high but not at low density. The deduced amino acid sequence of hensin shows that it is composed of three domains, SRCR, (complement subcomponents Clr/Cls, Lleg F, Bmp1) CUB, and (Zona Pellucida) ZP . SRCR domains are cysteine rich domains of unknown function found in a variety of proteins . The CUB domain was first identified in tolloid, a protein that activates decapentaplegic (dpp, the Drosophila TGFβ homologue) and mutations in that domain prevent dpp activation . The ZP domain, present in ZP sperm receptor proteins, bears some similarity to another TGFβ-binding protein, the type III receptor . Although these results suggest that hensin might bind to TGFβ or one of its many mammalian homologues, there is no evidence for this at present. Three other cDNAs have been reported that are composed of SRCR, CUB, and ZP domains. These include: CRP-ductin, a cDNA overexpressed in mouse intestinal crypt cells ; ebnerin, a partial cDNA overexpressed in rat von Ebner's gland ; and DMBT1, a large human sequence that resides in chromosome 10q25-26 is deleted in a substantial fraction of several malignant brain tumors such as gliomas and glioblastomas . This region has also been implicated in other epithelial cancers such as prostate cancer. Because these sequences were first identified in four different species and the presence of large number of proteins with one or more of these domains in each species, it was not possible to decide by simple sequence comparisons whether they represented a new gene family, or whether they were alternately spliced versions of the same gene. We cloned the mouse and rabbit genomic sequences of hensin and partial sequences of these two led to the conclusion that all four sequences are derived from the same gene (Takito, J., L. Yan, C. Hikita, S. Vijayakumar, D. Warburtar, and Q. Al-Awqati, manuscript submitted for publication). Hensin appears to be involved in terminal differentiation and it is well-known that interruption in terminal differentiation pathways often leads to cancer. These occurrences raise the intriguing possibility that hensin is a tumor suppressor. | Study | biomedical | en | 0.999997 |
10085302 | Tissues used in this study were stage XXI human fetal limbs, 50-d gestation, provided by the Central Laboratory for Human Embryology (University of Washington, Seattle, WA). Tissues were frozen in OCT compound (Miles Laboratories Inc.) and sectioned with cryostat. The sections (8–10 μm) were stored at −70°C until used. Probes specific for type IIA and IIB procollagen were used. A 207-bp cDNA, H-IIA, encoding exon 2 of human collagen type IIα1(II) was used to detect type IIA procollagen mRNA. Primers (5′ primer, 5′-CGTGAATTCCAGGAGGCTGGCAGCTGTGTG-3′; 3′ primer, 5′-GATGGATCCGGCGAGGTCAGTTGGGCAGAT-3′) that flank the exon 2 splice site were used to amplify a 207-bp fragment with EcoRI and BamHI restriction sites from 54-d human fetal embryonic tissue total RNA by using reverse transcription-polymerase chain reaction (RT-PCR), and cloned into pGEM-3zf(+) expression vector ( Promega Corp. ). This construct was used to generate antisense and sense riboprobes by in vitro transcription for in situ hybridization. Antisense 35 S-labeled RNA probe was transcribed by SP6 RNA polymerase on EcoRI linearized DNA template. Sense RNA probe was transcribed by T7 RNA polymerase on DNA template linearized with BamHI. The RNA transcripts were labeled with a 35 S-UTP ( New England Nuclear ). For detecting human type IIB procollagen mRNA, an oligonucleotide probe was used containing 12 nucleotides of exons 1 and 12 nucleotides of exon 3, 5′-CTCCTGGTTGCCGGACATCCTGGC-3′ . The probe was labeled with 5′-(a-thiol- 35 S)-ATP ( New England Nuclear ) using terminal deoxynucleotidyl transferase. In situ hybridization was performed as described previously . Three antibodies were used for immunohistochemistry of type II procollagen, and another two were used to detect BMP-2 and IGF-1 by ELISA and Western blots. Rabbit antisera against recombinant human type IIA-GST (IIA) only recognizes the exon 2 domain of type II procollagen . Rabbit antisera IIC reacts with bovine COOH-propeptide of type II collagen (provided by Dr. A. Robin Poole) and rat antisera against bovine type II collagen, IIF (provided by Dr. M. Cremer), recognizes the triple-helical domain of type II collagen. Preimmune sera from the rabbit producing anti–type IIA procollagen antibodies and nonimmune rat serum (Jackson ImmunoResearch Laboratories, Inc.) were used as controls. Anti–human integrin β1 mAb ( GIBCO BRL ) was used to demarcate the periphery of chondrocytes. TGF-β1 antibodies were obtained from Santa Cruz Biotechnology . They are specific for active TGF-β1. IGF-1 antiserum was from Austral Biologicals. The BMP-2/4 mAb (AbH3b2/17) was kindly provided by Dr. Elizabeth Morris (Genetics Institute, Cambridge, MA). This reagent, AbH3b2/17, was made by standard mAb procedures using full length recombinant human BMP-2 as the immunogen. It reacts with both BMP-2 and BMP-4. Details of antibody specificity have been described in Yoshikawa et al. and Bostrom et al. . Frozen sections (8–10 μm) mounted on polylysine coated slides ( Fisher Scientific Co. ) were fixed in 4% paraformaldehyde for 10 min at room temperature, and incubated with hyaluronidase (1 mg/ml) for 30 min at 37°C. Sections were blocked in PBS containing 10% (vol/vol) normal donkey serum (blocking buffer, Jackson ImmunoResearch Laboratories, Inc.) for 1 h at 37°C. All primary antibodies were diluted in PBS containing normal donkey serum (1% vol/vol). Antiserum IIA was used at a dilution of 1:400, IIC was 1:100, IIF was 1:50, and integrin β1 was 1:50. For double immunostaining, primary antibodies (IIA and IIF, IIC and IIF, or integrin β1 and IIF) were mixed well and incubated with sections overnight at 4°C. After washing in PBS, sections were incubated sequentially with appropriate secondary antibodies [cyanine 3 conjugated donkey anti–rabbit IgG F(ab′) fragment with a dilution of 1:200, FITC conjugated donkey anti–rat IgG F(ab′) fragment with a dilution of 1:100, or cyanine 3 conjugated donkey anti–mouse IgG F(ab′) fragment with a dilution of 1:200, Jackson ImmunoResearch Laboratories, Inc.] for 30 min at room temperature. Hoechst dye 33258 (1 μg/ml, Calbiochem-Novabiochem Corp. ) was used for fluorescent nuclear stain for 10 min at room temperature. After washing, sections were mounted in fluorescent mounting medium (Vector Laboratories, Inc.) and viewed on a Nikon Optiphot using DM445 (for Hoechst dye), DM510 (for FITC), and DM580 (for cyanine 3) filter cubes. Normal rabbit and rat serum were used as control instead of primary antibodies. Images were collected on a BioRad MRC600 scanning laser confocal microscope mounted on a Nikon Optiphot. Data were collected using either a Nikon 20×/0.50 or a 40×/0.70 NA dry objective. The BioRad A1-A2 cubes were used with an Argon laser producing excitation at 514 nm and collecting emission at 520–560 nm (green) and >600 nm (red). Optical sections were ∼2 μm with the 20× objective and 1 μm with the 40× objective. Full frame (768 × 512) 8-bit images were collected for analysis and overlaid in 24-bit RGB using Adobe Photoshop. High resolution images were collected on a Deltavision SA3.1 wide-field deconvolution optical sectioning device (Applied Precision, Inc.) mounted on an Olympus IMT-2 microscope. Data were collected using either a Nikon 60×/1.4 or 100×/1.4 NA objective using oil with an i.r. = 1.515. Hoechst dye 33258 (blue) was excited at 360/20 nm and emission collected at 457/25 nm. Fluorescein (green) was excited at 490/10 nm and emission collected at 528/19 nm. Cyanine 3 (red) was excited at 555/14 nm and emission collected at 617/36 nm. Optical sections were collected at 200 nm per step and deconvolved with a measured optical transform function per Sedat and Agard . Under these conditions we normally obtained 90 nm lateral and 400 nm axial resolution. Images were collected at 512 × 512 pixels at 12-bits/pixel. Final pixel depth is 16-bit. Images were exported as 24-bit TIFF images. The immunolocalization techniques used have been described previously . In brief, for en bloc localization of type IIA in fetal cartilage, samples were first exposed to chondroitinase ABC ( Sigma Chemical Co. ), 290 U/ml PBS for 2 h at 37°C, followed after rinsing by immersion overnight at 4°C in primary antibody (pAb IIA) diluted 1:5 in PBS. After a substantial wash in PBS, the samples were immersed in goat anti–rabbit 5-nm secondary gold conjugate ( Amersham Corp. ) diluted 1:3 in BSA, pH 7.8, overnight at 4°C. The samples were washed, fixed in aldehydes containing in 0.1% (wt/vol) tannic acid for 60 min followed by 1% OsO 4 for 120 min, then dehydrated and embedded in Spurr's epoxy. To further clarify the localization of type IIA procollagen NH 2 -propeptide within the fibrils, cartilage containing perichondrium from the same fetus was sheared in 0.2 M ammonium bicarbonate, pH 7.6, using an Omni International 2000 homogenizer. The homogenate was washed three times with resuspension in PBS and centrifugation at 600 g for 5 min. The resulting homogenate was either directly deposited onto carbon coated grids and stained with 3% phosphotungsic acid, pH 7.0, labeled only with primary antibody (1:5 in PBS) before staining, or labeled with primary antibody followed by secondary antibody 5-nm gold conjugate (1:3 in BSA) before staining. RCJ 3.1 C5.18 cells were maintained in α-MEM supplemented with 10% heat-inactivated FCS . The cells were labeled after the last medium change for 24 h in serum-free α-MEM (5 ml/dish) supplemented with 50 μg/ml ascorbate and 50 μg/ml β-aminoproprionitrile fumarate and containing 25 μCi/ml of [ 3 H]proline (>20 Ci/mmol, Amersham Corp. ) and 50 μCi/ml of [ 35 S]cysteine . After 24 h of culture, the medium was adjusted to 5 mM EDTA and 1 mM N -ethylmaleimide. Proteins were precipitated by the addition of 300 mg/ml of ammonium sulfate which was stirred overnight at 4°C. The precipitate was collected by centrifugation at 15,000 rpm at 4°C for 30 min in an SS34 rotor (Sorvall Instrument). The precipitate was suspended in 1 ml PBS and then dialyzed for 48 h against the same buffer. The total RNA was extracted from RCJ 3.1 C5.18 cells by TRIZOL Reagent ( GIBCO BRL ) following the manufacturer's instructions. RT-PCR was used to identify type IIA and IIB mRNA. Two primers, 5′-TCGGGGCTCCCCAGTCGCTGGTG-3′ (exon 1) and 5′-GATGGAGAACCTGGTACCCCTGGA-3′ (exon 7), were used to amplify type IIA and IIB cDNA fragments which are 457 and 253 bp, respectively. PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. To identify type II procollagens, the proteins collected from culture medium were separated on 5% SDS-polyacrylamide gel and then analyzed by Western blotting. Three antibodies, rabbit anti-IIA + GST (IIA) at 1:1,000 dilution, rat anti-IIF at 1:500, and rabbit anti-IIC at 1:1,000, were used. Anti–rabbit and –rat IgG conjugated with HRP (Jackson ImmunoResearch Laboratories, Inc.) were applied and detected by SuperSigal ® Chemiluminescent Substrate ( Pierce Chemical Co. ). Pepsin solubilized chick type II collagen ( Sigma Chemical Co. ) was used to indicate the migration of the type II collagen α chain. RT-PCR was carried out to amplify a 315-bp fragment encoding the entire common domain of the type II collagen NH 2 -propeptide from exon 3 (beginning of minor helix) through exon 8 (beginning of the major helix) from 54-d human fetal embryonic tissue total RNA. The forward 35-mer primer was 5′-AATGGATCCCAACCAGGACCAAAGGGACAGAAAGG-3′. The reverse 29-mer primer was 5′-ATATGCGGCCGCCATTGGTCCTTGCATTACTCCCAACTGGGC-3′. PCR products were digested with BamHI and NotI, and cloned into a pGEX-4T-2 vector ( Pharmacia Biotech, Inc. ). cDNA sequencing was used to confirm the correct reading frame. The expression and purification of the recombinant human type II collagen NH 2 -propeptide (rhIIN-GST, exons 3–8) was carried out by Bulk and RediPack GST purification modules ( Pharmacia Biotech, Inc. ) following the manufacturer's instructions. The fusion protein (rhIIN-GST) was analyzed by rabbit anti–IIA + GST antibody or goat anti-GST antibody ( Pharmacia Biotech, Inc. ) on Western blotting. 60 nM recombinant human type IIA procollagen NH 2 -propeptide , 60 nM human IGF-1 (R&D Systems), or 15 nM human BMP-2 (Genetics Institute) was incubated for 1 h at room temperature in 1 ml of PBS containing 1 mM CaCl 2 , 3 mM MgCl 2 , and 1 mg/ml BSA. 10 μl rabbit antisera against NH 2 -propeptide or preimmune serum was added to the samples and incubated for 2 h at 4°C. 20 μl of protein A–Sepharose beads ( Pharmacia Biotech ) were added and incubated for 3 h. Beads were pelleted for 1 min and precipitated immune complexes were washed five times with 1 ml PBS, pH 7.2, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and once with 1 ml of 10 mM Tris-HCl, pH 6.8. The samples were resuspended in 40 μl Laemmli sample buffer (without DTT), boiled for 5 min, electrophoresed through SDS polyacrylamide gels under nonreducing conditions, and electroblotted onto PVDF membranes. The membranes were blocked with 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20 containing 3% BSA, and incubated in the same buffer for 1 h at room temperature with primary antibody, anti–human BMP monoclonal or anti–human IGF-1 monoclonal (Austral Biologicals), both at a dilution of 1:500. Anti–mouse secondary antibodies were used and detected by Western blue stabilized substrate for alkaline phosphatase ( Promega Corp. ). For comparison of binding to IIA and IIB procollagens, recombinant proteins for type IIA NH 2 -propeptide (rhIIA-GST) or II NH 2 -propeptide (rhIIN-GST, exons 3-8 of the NH 2 -propeptide) were mixed with BMP-2 as above and immunoprecipitated with BMP specific antiserum. Immunoprecipitates were separated by electrophoresis on a 15% SDS polyacrylamide gel, transferred to PVDF membranes, and reacted with antiserum to type IIA-GST. To test whether BMP-2 binds to natural type IIA procollagen, the 3 H- and 35 S-labeled proteins collected from C5.18 cell medium were immunoprecipitated with BMP-2 antibody. In brief, 100 μl of labeled proteins diluted in NET-buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, pH 8.0, and 0.25% gelatin) to 1 ml was mixed with 10 μl of mouse serum–agarose ( Sigma Chemical Co. ) for 1 h at 0°C. Mouse serum– agarose was discharged after centrifugation. 200 ng of BMP-2 was added to the supernatant and incubated for 1 h at 4°C, then 5 μl of BMP-2 antibody was applied and incubated an additional 1 h at 4°C. After incubation, 20 μl of protein A–Sepharose beads ( Pharmacia Biotech, Inc. ) was added and incubated for 1 h at 4°C. Beads were pelleted for 1 min and the precipitated immunocomplexes were washed three times with 1 ml NET-buffer. The samples were resuspended in 30 μl Laemmli sample buffer and boiled 5 min. Normal mouse serum was used as negative control, instead of BMP-2 antibody. The type IIA procollagen and type II collagens were immunoprecipitated by rabbit antiserum to type IIA-GST and rat antiserum against the fibrillar domain of type II collagen. Labeled proteins were visualized by autoradiography after separation on 5% SDS polyacrylamide gel using Amplify (Nycomed Amersham Inc. ). 96-well flat bottomed plates were coated overnight at 4°C with 5 or 10 ng/well TGF-β1, BMP-2, bFGF, IGF-1, and GST in 0.1 M Tris-HCl, 50 mM NaCl, pH 7.4 (Tris-NaCl), respectively. Plates were washed three times with PBS, pH 7.2, containing 0.1% (vol/vol) Tween 20 (PBS/Tween). To block nonspecific binding, plates were incubated for 1 h at 20°C with PBS/Tween containing 3% (wt/ vol) BSA and washed four times in PBS/Tween. Dilutions of rhIIA-GST fusion protein and GST , from 1 to 5,000 ng/well, in Tris-NaCl were added to the coated wells and incubated at 37°C for 2 h. Plates were washed five times with PBS/Tween. Plates were incubated for 4 h with PBS/Tween/BSA buffer, then incubated for 2 h at 20°C with a 1:1,000 dilution of anti–IIA-GST antibodies in PBS/Tween. Plates were washed five times with PBS/Tween and incubated for 2 h at 20°C with a 1:5,000 dilution of goat anti–rabbit IgG-alkaline phosphatase conjugate in PBS/Tween and washed five times with PBS/Tween. Plates were incubated for 30–60 min with 3 mM p -nitro-phenylphosphate substrate in 0.05 M Na 2 CO 3 and 0.05 mM MgCl 2 buffer, and absorbance was measured at 405 nm using a Hewlett Packard ELISA microplate reader. In addition, the substrates and ligands were reversed. rhIIA-GST fusion protein or IIA protein (only exon 2) alone was plated at 10 ng/well. BMP-2 and mAb against rhBMP-2 were incubated sequentially as above. Then, secondary antibody and color reactive substrate were used to detect the binding. Each data point was in duplicate from three independent experiments. To determine whether type IIA procollagen is involved in early stages of chondrogenesis, we investigated the specific localization of the NH 2 -propeptide before and during chondrogenesis. In the developing limb, distal skeletal structures differentiate later than proximal structures . Therefore, 50-d human embryonic limb tissue was used because many stages of chondrogenesis can be observed. Antibodies specific for different domains of the collagen molecule were used to localize the IIA NH 2 -propeptide, COOH-propeptide, and triple-helical (fibrillar) domains of type II procollagen. RNA probes were used to confirm the distribution of mRNA. The approximate locations of epitopes and mRNA probes are shown in Fig. 1 . Double immunofluorescence was performed on tissue sections using the triple-helical antibody together with either the NH 2 - or COOH-propeptide–specific primary antibodies and fluorescent secondary antibodies. Fluorescence was visualized by confocal laser-scanning microscopy . In the condensing mesenchyme of the emerging digital rays, signal for type IIA NH 2 -propeptide can be observed colocalized with the triple-helical domain . At this time, the cells are closely packed condensations and there is no evidence of chondrocyte-characteristic morphology. In serial sections, mRNA levels are below the level of detection with routine in situ hybridization. However, the more sensitive immunolocalization identifies these cells as the site of future cartilage differentiation. More proximal in the developing radius, different stages of chondrogenesis are present. D–F in Fig. 2 show the distribution of type IIA and IIB procollagen mRNA splice forms. Type IIA collagen mRNA is synthesized by chondroprogenitor (CP) cells and type IIB collagen by chondroblasts and chondrocytes (C). In chondroprogenitor tissue, where only type IIA procollagen mRNA is detected, both NH 2 -propeptide and triple-helical domains are colocalized . There is a gradient of distribution of type IIA NH2-propeptide with the greatest immunoreactivity in the chondroprogenitor zone. The gradient distribution of fibrillar domain in H exceeds the range of sensitivity of the detector. Consequently, the green fluorescence in the CP region is underrepresented to reduce blurring due to the high signal in the C region. In the chondroblasts and chondrocytes, where type IIB mRNA is detected, the NH 2 -propeptide can still be visualized in the ECM . In contrast to the NH 2 -propeptide, double immunofluorescence using antibodies to the COOH-propeptide and triple-helical domains reveals a different pattern of fluorescence . The COOH-propeptide is not colocalized with the triple-helical domains in the ECM, but appears to be localized inside the cells . To define more precisely the localization the type II procollagen domains during chondrogenesis, tissue sections were visualized using Delta Vision™ microscopy. The Delta Vision™ system utilizes broad field optics coupled with computerized deconvolution of the optical image using Fourier transformation. A Z-stack of optical sections through 3.2 μm can be viewed with a resolution of ∼90 nm. Selected fields representing stages of chondrogenesis shown in Fig. 2 are presented in Fig. 3 . In addition to the immunolabeling of collagen domains shown above in confocal micrographs, the fluorescent dye Hoescht 35258 can be used to identify nuclei. Immunoreactivity of the NH 2 -propeptide (red) and fibrillar domains (green) merged images are shown. Independent visualization of single fluorescence confirmed localization of both Cy3 (red) and FITC (green) in regions that appear orange or orange-red. As shown above, chondroprogenitor cells synthesize type IIA procollagen mRNA while more mature chondrocytes synthesize type IIB procollagen. In the chondroprogenitor tissue, the cells are tightly packed with large nuclei, little cytoplasm, and very little ECM is observed. However, the small amount of staining around the cells can clearly be seen in this merged image to be reddish yellow indicating colocalization of NH 2 -propeptide and the triple-helical domains. In chondroblasts , an accumulation of type IIA NH 2 -propeptide and fibrillar collagen can be observed. Less mature cells are in the upper left half of the photograph while the more mature chondrocytes are in the lower right half of the photograph. In the zone of mature chondrocytes , the cells are even larger and contain distinct secretory granules lying close to the nucleus. More cellular detail in these rounded cells can now be resolved. In the ECM, reddish orange-staining areas of propeptide are localized in the interterritorial matrix where it has been displaced by newly synthesized type IIB procollagen (green). In previous studies and shown above, in situ hybridization to mRNA demonstrated that these chondrocytes transcribe only type IIB procollagen mRNA and no longer synthesize the type IIA NH 2 -propeptide. The newly synthesized type IIB procollagen can be seen in the secretory granules surrounding the nucleus and deposited immediately around the cell. Fig. 3 D shows the hypertrophic zone where streaks of type IIA procollagen remain in a matrix that contains primarily type IIB collagen. To further confirm the extracellular localization of the NH 2 -propeptide, serial sections were stained with antibodies to type II procollagen COOH-propeptide, type II triple-helical domain, and integrin β1 . In Fig. 3 (E and F), the double immunohistochemistry with anti–COOH-propeptide and anti-helical domain antibodies is shown. Note that only the triple-helical domain (green) is deposited into the ECM of chondroprogenitor cells while the COOH-propeptide (red) is colocalized with the triple-helical domain in the secretory granules or alone (red). The intercellular structures staining with the COOH-propeptide antiserum is currently under investigation. Most of these structures do not react with the Golgi apparatus or endoplasmic reticulum characteristic antibodies, such as anti-Golgi 58K protein and anti-Hsp47, respectively (data not shown). Preimmune serum used as the primary antiserum is shown as a negative control and the cell periphery was confirmed by localization of integrin β1 . The yellow signal indicates that integrin β1 is colocalized with type II collagen triple-helical domains . To determine the molecular organization of the NH 2 -propeptide, localization of type IIA procollagen in embryonic chondrogenic tissue was performed and visualized using electron microscopy. Antiserum to the NH 2 -propeptide was used to localize the procollagen in tissue . The results demonstrate localization of antibody-bound gold particles on the surface of collagen fibrils present in perichondrial tissue. The fibrils shown here also react with the type II collagen helical domain antibody. To further clarify the position of the NH 2 -propeptide within the fibrils, individual fibrils were released from tissue matrix by shearing in ammonium bicarbonate buffer using a tissue homogenizer , incubated only with type IIA specific antibody , then further incubated with 5-nm gold secondary antibody conjugate . Before antibody treatment, the fibrils have an irregular surface and the periodic banding pattern of type II collagen characterized by Eikenberry et al. . After incubation with type IIA antibody, protrusions from the fibril surface can be seen . The identity of the protrusions as primary antibody is confirmed by secondary antibody–gold conjugate . A determination of periodicity following gold conjugate is complicated by the additional length of the complex (primary antibody–secondary antibody–gold particles) and by some secondary antibodies carrying more than one gold particulate. Therefore, the estimate of antigen spacing was made from the primary antiserum photomicrographs. Taken together, these results indicate that the NH 2 -propeptide is present at the surface of the type II collagen fibril and found at locations corresponding to the periodic repeat of the collagen molecule. The presence of type IIA NH 2 -propeptide in ECM of chondroprogenitor cells suggests that it has a function before differentiation of the chondrocyte and could play a role in the induction of chondrogenesis. To assay for binding, immunoprecipitation of BMP-2 and IGF-1 with IIA NH 2 -propeptide antibody was performed. rhIIA-GST protein isolated from the recombinant GST fusion protein was used. rhIIA (60 nM), human recombinant BMP-2 (15 nM), or IGF-1 (60 nM) was incubated for 1 h at room temperature in 1 ml of PBS binding buffer, immunoprecipitated with anti-IIA NH 2 -propeptide antibody, and the amount of BMP-2 or IGF-1 bound to rhIIA protein was detected on Western blots with monoclonal anti–BMP-2 or IGF-1 antibody. As shown in Fig. 5 A (lane 1) BMP-2 can be immunoprecipitated by IIA NH 2 -propeptide antiserum. Control reactions show no immunoprecipitation with BMP-2 alone and no immunoprecipitation of the BMP-2–rhIIA protein complex with preimmune serum . No immunoreactivity for IGF-1 was detected when a mixture of IGF-1 and exon 2 protein was immunoprecipitated with NH 2 -propeptide antiserum . To determine whether BMP-2 binding was specific for the type IIA splice form of type II collagen, binding of BMP-2 to recombinant type IIA (rhIIA, exon 2) was compared with binding to recombinant type IIB NH 2 -propeptide . Immunoprecipitation was performed by mixing 4.0 μg human recombinant type IIA fusion protein (rhIIA-GST), and 1.0 μg BMP-2 or human recombinant type IIB NH 2 -propeptide (rhIIN-GST), and BMP-2 and precipitating with antibody to BMP-2. Western blot analysis was performed and recombinant type IIA fusion protein identified with specific antiserum. Type IIA (rhIIA-GST) was immunoprecipitated with antiserum to BMP , but recombinant type IIB NH 2 -propeptide nor GST could be immunoprecipitated. Fig. 5 B, lanes 4–6, shows that antisera against rhIIA-GST can react with rhIIA (exon 2), rhIIN-GST (exons 3–8), and GST when they are run on the gel. Media from C5.18 cultured chondroblasts was used to demonstrate binding of natural type IIA procollagen to BMP-2. Fig. 6 A shows that cells express mRNA for both type IIA and IIB procollagens. Protein products were separated on a 5% SDS-polyacrylamide gel and transferred to PVDF membrane for Western blot analysis of type II collagens . Lanes 1 and 3 show immunoreactivity with the type IIA NH 2 -propeptide antiserum and type II COOH-propeptide antiserum, identifying this band as pNC type IIA procollagen, shown previously for human cells . Antiserum to the fibrillar domain of type II collagen indicates the presence of multiple forms of type II collagen in the medium . These forms include type IIA pNC procollagen, type IIB pNC procollagen, type II pC procollagen, and mature α chains . Pepsin solubilized type II collagen α chain is shown in Fig. 6 B, lane 4. Specific antisera were used to precipitate procollagens from the medium, type IIA procollagen , and all type II collagens . When recombinant BMP-2 was added to the medium and proteins immunoprecipitated with BMP-2 antibody, type IIA procollagen alone was observed . To estimate the strength of interaction between NH 2 -propeptide and BMP-2, the binding of various growth factors to alternatively spliced type IIA procollagen NH 2 -propeptide domain (rhIIA) expressed as a GST-fusion protein was tested. The growth factors bFGF, IGF-1, BMP-2, and TGF-β1, all known to be involved in chondrogenesis, were tested in a solid phase binding assay. Fig. 7 A shows the results of binding of rhIIA-GST to immobile BMP-2, bFGF, and IGF-1. rhIIA-GST was added in increasing concentrations and the amount bound was measured with antiserum to NH 2 -propeptide. No binding of rhIIA-GST was observed with bFGF and IGF-1 up to 10 μg/well . Similar results were observed with TGF-β1 . Similar results were also obtained when substrates and ligands were reversed, i.e., rhIIA-GST was coated on plates and exposed to BMP-2. Antibody to BMP-2 was used to detect binding (data not shown). Scatchard plot analysis of the interaction indicated a K D of 7.65 nM for TGF-β1 and 5.23 nM for BMP-2. The mechanism of induction and differentiation of the skeleton represents a basic developmental question and thus has attracted a great deal of attention. Substantial progress has been made in clarifying the roles of patterning genes such as pax, hox, hedgehogs, FGFs, genes that induce musculoskeletal cell phenotypes such as the Myo D family of transcription factors, and the extracellular signaling factors, BMPs. The findings presented here indicate that type IIA procollagen could potentially play a role in induction and differentiation of the skeleton. Type IIA procollagen is synthesized by chondroprogenitor cells and deposited into the ECM. It retains the NH 2 -propeptide, but the COOH-propeptide is removed. The NH 2 -propeptide of type IIA procollagen binds to BMP-2 and TGF-β1, factors present in the tissue and known to induce chondrogenesis in vivo and in vitro , respectively . These results show for the first time that type IIA pN-procollagen is deposited into the ECM and suggest a novel function for the collagen NH 2 -propeptide. Type IIA procollagen is the predominant form of type II collagen in chondroprogenitor tissue and remains in the tissue after cells switch synthesis to type IIB collagen. Over time however, the predominant collagen becomes type IIB collagen, and the type IIA procollagen is removed. We do not know what enzymes are involved in type IIA procollagen turnover or whether the NH 2 -propeptide alone is cleaved from the collagen fibril, although the NH 2 -propeptide can be cleaved by stromelysin, which cleaves between the N-protease cleavage site and the beginning of the major triple helix , an enzyme known to be increased in hypertrophic cartilage (Zhu, Y., and L.J. Sandell, unpublished observations) and the collagen N-protease which cleaves 8 amino acids downstream of the minor triple helix of the propeptide . Piccolo et al. have shown recently that the chordin–BMP-4 complex is proteolytically processed in chordin by the matrix metalloprotease xolloid, thereby releasing active BMP-4. Cleavage of chordin alone inhibits its ability to bind BMP-4. A similar cleavage mechanism by a related enzyme, tolloid, occurs in the sog–dpp complex . For type IIA procollagen, an analogous cleavage mechanism may exist, as both N-protease and stromelysin are members of the same class of astacin proteases as tolloid and xolloid. The data presented here suggest that BMPs may be localized to sites of chondrogenesis by direct interaction with the NH 2 -propeptide of type IIA procollagen. Support for this hypothesis is derived from a similar interaction of chordin and sog, homologues of the NH 2 -propeptide, with BMP-4 and decapentaplegic . The interactions regulate presentation of the morphogen to the cell. The homology between sog, chordin, and NH 2 -propeptide includes placement of 10 cysteines, conserved across types I, IIA, III, and α2 (V) collagens and thrombospondin, and placement of amino acids glycine, tyrosine, tryptophane, proline, glycine, and proline at residues 38, 41, 47, 82, 84, and 92 of the type IIA procollagen NH 2 -propeptide. Although we have not directly compared the binding of chordin or sog with type IIA NH 2 -propeptide in the same assay system, we can compare the estimated K D for the binding of BMP-4 to chordin (3 × 10 −10 M) to the estimated K D for type II NH 2 -propeptide binding to BMP-2 and TGF-β1 (5–7 × 10 −9 M). These values compare favorably with the binding of BMPs to their receptors, 9 × 10 −10 M for Xenopus BMP2/4 receptor , 2.5 × 10 −10 M for thick veins dpp receptor , and a range of 2 × 10 −10 to 3.5 × 10 −9 M for binding of BMPs to various cell receptors . Another protein, noggin, can bind to BMP-4 and functions similarly to chordin in dorsal– ventral patterning and neural induction. Noggin is a member of a new family of BMP-binding proteins which includes gremlin in neural crest, the head-inducing factor cerberus, and the tumor suppressor DAN . Although their binding affinities for BMP-4 are different (2 × 10 −11 M for noggin and 3 × 10 −10 M for chordin) both proteins are able to dorsalize mesoderm at 1 nM in Xenopus embryos . Recently, noggin has been shown to be involved in chondrogenesis. That is, in noggin-deficient mice, among other central nervous system and somite patterning defects, cartilage condensations initiate normally but develop hyperplasia, and development of joints in the limb fails . The involvement of noggin in chondrogenesis is intriguing, and the relationship between noggin and type IIA collagen NH 2 -propeptide binding to BMP is unknown. However, the expression pattern of noggin is quite different from type IIA procollagen and more closely resembles the type IIB procollagen splice form . Consequently, its role in chondrogenesis is likely to be different from type IIA procollagen. The primary sequence of noggin is not homologous to type IIA NH 2 -propeptide. Reddi and colleagues have investigated the binding of ECM proteins to TGF-β and bone morphogenetic proteins. They have shown that TGF-β, BMP-3 , and BMP-7 bind avidly to type IV collagen, and to a lesser extent, types I, VI, and IX collagens and heparin. They do not bind to types II, III, V, or X collagens, laminin, fibronectin, or proteoglycans . Consistent with these results, we show the fibrillar domain of type II collagen does not bind to BMP-2. In general, only relative binding affinities were reported. However, the K D of BMP-7 and type IV collagen was estimated to be 5 × 10 −11 M . The localization of type IIA procollagen shown here is consistent with a role for propeptide in regulating the distribution of BMPs. This localization could potentially apply in four primary, but distinct processes. The first is the localization of type IIA procollagen at epithelial–mesenchymal boundaries. Wood et al. immunolocalized type II collagen and we and others have shown that these cells synthesize predominantly type IIA mRNA. Lui et al. showed type II collagen mRNA is initially synthesized by neuroepithelial cells, then by both epithelial and mesenchymal cells, then only mesenchymal cells. The mesenchymal cells proceed to chondrogenesis because they express the receptors necessary to respond to the inducing agent. Secondly, type IIA procollagen is localized in prechondrogenic condensations before differentiation into chondrocytes, as shown above. Thirdly, type IIA procollagen is transiently expressed in other areas where BMPs are involved in induction of differentiation and could be involved as nonchondrogenic processes. For example, type IIA procollagen mRNA has been found in early kidney development, skin before terminal differentiation of keratinocytes, developing aorta, lung buds, salivary gland, adrenal cortex, notochord, somites, and apical ectodermal ridge in mice and in humans . Fourthly, type IIA is present in periosteum and perichondrium, predominant sites of ectopic bone formation. The mechanism of BMP induction of mesenchymal cells after binding and localization by type IIA procollagen remains to be clarified. It is possible that IIA-bound BMPs could induce chondrogenesis. On the other hand, the NH 2 -propeptide–BMP complex could be liberated by an amino propeptidase or stromelysin, both known to be able to cleave the propeptide when these enzymes become available in the ECM. Lastly, the NH 2 -propeptide–BMP complex could be disengaged, releasing BMP to bind to the cellular receptor. Piccolo et al. have hypothesized that chordin inactivates potential binding of BMP-2 to the cellular receptors, based on inhibition of BMP-2 stimulation of osteogenesis in C3H10T1/2 cells. While the binding mechanism between the chordin– BMP-4 and NH 2 -propeptide–BMP-2 complexes may be similar, the functional outcome may be quite distinct. Chordin is synthesized and secreted as a soluble protein, while type IIA procollagen is deposited into the ECM. The NH2-propeptide can remain attached to the triple-helical domain or be liberated by cleavage. Chordin is thought to function by removing BMP-4 from the site of potential inductive activity, in this case inducing ventralization in Xenopus . A similar interaction occurs in the dorsal ventral patterning in Drosophila . That is, the sog , a homologue of type IIA NH 2 -propeptide and chordin , functions as an antagonist of decapentaplegic, a member of the TGF-β superfamily . The similar functional outcome of interactions of chordin–BMP-4 and sog–decapentaplegic establishes a conserved mechanism for dorsal–ventral patterning that is shared by vertebrates and arthropods . The binding of type IIA NH 2 -propeptide to BMPs suggests a novel function for this protein domain. We show that type IIA procollagen is synthesized and deposited into the ECM. This fibrillar domain of the collagen could then provide a substrate for mesenchymal cells while the NH 2 -propeptide localizes the protein capable of inducing chondrogenesis. As the cells differentiate into chondrocytes, exon 2 encoding the NH 2 -propeptide is removed by alternative splicing of the mRNA. Consequently, by controlling the availability of NH 2 -propeptide, a mechanism is built in to control the amount of morphogenetic agent the cells are exposed to. Subsequently, type IIA procollagen is synthesized in the perichondrium and periosteum where it can help establish a reservoir of BMP. Pattern induction, whether in early body axis or elements of the skeletal system, is thus guided by the result of a gradient of morphogen bound to a specific protein domain. We propose that the interactions of sog, chordin, and type IIA procollagen NH 2 -propeptide with members of the TGF-β superfamily represent a biological paradigm whereby the presentation of morphogenetic proteins can be regulated. | Study | biomedical | en | 0.999994 |
10087255 | Membrane wash buffer (MWB) consisted of 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl 2 , 50 mM Hepes, pH 8.0, 1 mM dithiothreitol, 0.5 mM ATP, 1 μg/ml aprotinin, and 1 μg/ml leupeptin. Sonication buffer consisted of 50 mM NaPO 4 , pH 8.0, and 300 mM NaCl. Protease inhibitors benzamidine (5 mM final concentration; Sigma Chemical Co. ), PMSF (0.5 mM final concentration; Sigma Chemical Co. ), and pepstatin A (1 μg/ml final concentration; Sigma Chemical Co. ) were included in the sonication buffer during sonication, but were not added in subsequent steps. Stop buffer consisted of 80 mM Tris-HCl, pH 8.0, 8 mM EDTA, 0.13% phosphoric acid, 10% Ficoll, 5% SDS, and 0.2% Bromophenol blue. TBS consisted of 100 mM Tris-HCl, pH 7.5, plus 0.9% (wt/vol) NaCl. WGA ( Sigma Chemical Co. ) was kept frozen as a 10 mg/ml stock at −80°C. Aphidicolin was kept as a 2.5 mg/ml stock in DMSO at −20°C. Membrane and cytosol fractions were prepared from unactivated Xenopus eggs as previously described . Demembranated Xenopus sperm chromatin was also prepared as previously described . Chromatin, at a final concentration of ∼40,000 sperm/μl, was stored at −80°C. For nuclear assembly reactions, 2 μl membranes (∼30 mg protein/ml), 20 μl cytosol (∼25–30 mg protein/ml, supplemented with 10 mM phosphocreatine, 1 mM ATP, and 50 μg/ml creatine phosphokinase as an ATP regenerating system), and 1 μl demembranated sperm chromatin were mixed on ice and transferred to 22–24°C to initiate nuclear assembly. All reactions were done using components that had been frozen and thawed once. For reactions that contained recombinant LAP2 fragments, 1 μl of LAP2 protein (in MWB) was added to mixed cytosol and membranes to yield the indicated final concentration of LAP2 fragment. Chromatin was added, and reactions were mixed again and transferred to 22– 24°C to initiate assembly. To assay for nuclear import, rhodamine-labeled nucleoplasmin was prepared according to Newmeyer et al. , and added to nuclei after 2 h of assembly in the presence or absence of LAP2 fragment 1–408. Nuclei were imaged by epifluorescence microscopy 30 min later (time = 2.5 h). As a negative control for import, WGA (final concentration, 1 mg/ ml) was added 5 min before adding fluorescent nucleoplasmin. WGA inhibits active transport by binding to O -GlcNAc-modified nucleoporins at the NPC . Escherichia coli cells, strain BL21(DE3)pLysS (Novagen, Inc.), were transformed with the pET-23a expression vector (Novagen, Inc.) containing inserts coding for either residues 1–408 or residues 1–187 of human LAP2β, or residues 1–164 of Xenopus LAP2 (see below). We followed the convention of numbering amino acids that excludes the initiating methionine, consistent with previous papers . The pET-23a expression vector adds a His tag (Leu-Glu-His 6 ) to the COOH terminus of the expressed protein. Thus, LAP2β fragment 1–408 is a 417–amino acid, 46.49-kD protein, and LAP2 fragment 1–187 is a 196–amino acid, 21.716-kD protein. To produce each recombinant protein, an overnight culture of a single colony was diluted 1:60 in fresh media. Upon reaching an OD 600 of ∼0.6, protein expression was induced with 0.4 mM isopropyl-β- d -thiogalactopyranoside (IPTG) for 3 h, and the bacteria were pelleted by centrifugation (6,000 g for 15 min at 4°C). The pellet was frozen in liquid N 2 and stored at −80°C. To purify each recombinant protein, the pellet was resuspended in sonication buffer, subjected to pulse sonification, and centrifuged (20,000 g for 20 min at 4°C). The supernatant was applied to a Ni-NTA–agarose column (Qiagen, Inc.), which was washed successively with 10 column volumes each of sonication buffer and sonication buffer plus 10 mM imidazole. Recombinant His-tagged proteins were eluted with sonication buffer containing 100 mM imidazole. The proteins were concentrated and desalted using Centricon-30 units (Amicon, Inc.). Small aliquots were frozen in liquid N 2 and stored at −80°C; the thawed proteins were stable at 4°C for at least a week. Mock purifications were done in parallel using uninduced bacteria to provide a control for potential nonspecific effects due to either imidazole or bacterial proteins. However, in no case did the effect of mock-purified proteins differ from that of buffer alone. Aliquots of assembly reactions were fixed in MWB containing 3.7% formaldehyde, 20 μg/ml Hoechst 33342 (a DNA stain; Calbiochem Corp. ). The samples were observed using a Nikon Microphot fluorescence microscope and photographed with Kodak Tri-X Pan 400 film. In some cases samples were imaged using a Photometrics SenSys cooled CCD camera, and images were processed and printed using IPLabSpectrum software. Samples for electron microscopy were fixed for 30 min on ice in 1.5% (vol/ vol) glutaraldehyde and 1% (vol/vol) paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. Samples were pelleted for 1 min in an Eppendorf centrifuge at 4°C, and the chromatin/nuclear pellet was rinsed in cacodylate buffer. Pellets were postfixed for 30 min at 4°C in 1% reduced osmium tetroxide, dehydrated, and embedded in Spurr's medium. Samples were sectioned (90-nm sections) and poststained in uranyl acetate followed by lead citrate. Electron micrographs of thin sections were taken on a TEM10 microscope ( Carl Zeiss, Inc. ) at 60 or 80 kV. DNA replication was assayed by incorporation of α[ 32 P]dCTP . In brief, 1 μl of α[ 32 P]dCTP (Redivue, 3,000 Ci/mmol; Nycomed Amersham ) was added to 24-μl nuclear assembly reactions (20 μl cytosol, 2 μl membranes, 1 μl chromatin [stock concentration ∼40,000/μl], plus 1 μl buffer or recombinant LAP2β polypeptides). As a negative control, aphidicolin was added at a final concentration of 50 μM; this agent inhibits the activity of DNA polymerase α. Alternatively, independent negative control reactions were made 1 mM in GTPγS to inhibit nuclear membrane formation and, indirectly, DNA replication. After 3 h, samples were combined with an equal volume of stop buffer, proteinase K ( Boehringer Mannheim GmbH ) was added to 1 mg/ ml final concentration, and samples were incubated at 37°C for 2 h. To detect incorporated α[ 32 P]dCTP, the protease-digested samples were mixed thoroughly by pipetting to ensure homogeneity, and 5-μl aliquots were electrophoresed through 0.8% agarose gels. Note that all reactions contained equal numbers of nuclei, and samples were processed without pelleting steps that might cause loss of material. Gel loading was monitored by ethidium stain contained in the gel. Dried gels were exposed to x-ray film, and signals quantitated with scanning densitometry using the Microcomputing Imaging Device (Imaging Research Inc.). In one experiment, the signal was quantitated by both phosphorimaging and densitometry, with the same results. To measure the time course of replication, 10-μl aliquots were removed every 15 min from 200-μl reactions (190 μl crude extract, 10 μl chromatin, 5 μl [ 32 P]dCTP, plus 6 μl of buffer or purified LAP2 fragment 1–408), digested with proteinase K, and the DNA was separated on agarose gels and quantitated by PhosphorImager as described above. Density substitution experiments were done essentially as described by Hua and Newport , using both freshly prepared crude nuclear assembly extracts (10,000 g cytoplasmic fraction), and high speed fractionated, frozen, reconstituted extracts, with the same results. Nuclei were assembled for 90–120 min in the presence or absence of 3.3 μM fragment 1–408. Crude reactions contained 60 μl cytoplasm with 2,000 sperm per μl, plus 0.5 mM BrdU ( Sigma Chemical Co. ), 0.5 mM MgCl 2 , and 0.2 μCi of α[ 32 P]dCTP per μl of extract. Fractionated/reconstituted reactions contained 60 μl cytosol, 12 μl membranes, and a total of 100,000 sperm chromatin, plus 0.2 μCi/μl α[ 32 P]dCTP and 0.5 mM BrdU. Reactions were stopped by adding 1 ml ice cold buffer A (50 mM KCl, 50 mM Hepes-KOH, pH 7.4, 5 mM MgCl 2 , and 1 mM DTT), incubated on ice for 5 min, centrifuged 5 min at 16,000 g in a microfuge, resuspended in 100 μl buffer A, made 0.5% in SDS and 0.4 mg/ml in proteinase K, and digested for 2 h at 37°C. DNA was then extracted three times with phenol-chloroform, once with chloroform, and ethanol precipitated using 0.3 M sodium acetate. Each ethanol pellet was resuspended in 100 μl TE, mixed with 12.7 ml of 1.75 g/ml CsCl, loaded into a Beckman 16 × 76 mm Quick-Seal Tube, and centrifuged 45 h at 30,000 rpm at 20°C in a Beckman Ti-70.1 rotor. 46– 50 fractions (250–300 μl each) were collected by needle puncture from the bottom of each tube. To quantitate radioactivity, an aliquot of each fraction was counted by liquid scintillation . The refractive index of each fraction was measured using a refractometer (Bausch & Lomb Inc.), and converted to density using the equation: d (in units of g/ml) = 0.99823 × d 20 20 , where d 20 20 is the specific gravity of solution at 20°C. Values for d 20 20 for cesium chloride at each index of refraction (n) are provided in the CRC Handbook . To detect lamin accumulation by immunoblotting, nuclei were assembled for 3 h in the presence or absence of human LAP2β fragments, diluted with 100 μl MWB, and then pelleted at top speed in an Eppifuge for 1 min and washed with 200 μl MWB. The washed nuclear pellet was resuspended in SDS-sample buffer, subjected to SDS-PAGE (12% gel), and proteins transferred to Immobilon PVDF membrane ( Millipore Corp. ). The Immobilon was blocked with 5% dry milk in TBS 0.1% Tween-20 (TBS-Tw) for 30 min, rinsed briefly in TBS-Tw, and incubated overnight at 4°C with either of two mAbs: antibody 46F7 , which is specific for Xenopus lamin B3 (formerly known as lamin L iii ), the major lamin found in Xenopus eggs , or a monoclonal directed against human lamin B ( Calbiochem ; final concentration 100 μg/ml in TBS-Tw). Blots were rinsed six times (5 min each) with TBS-Tw and then incubated for 1 h at 22–24°C with HRP-conjugated anti–mouse secondary antibody in TBS-Tw (Nycomed Amersham ). The blots were washed again (six times for 5 min) and developed using enhanced chemiluminescence (ECL) reagents (Nycomed Amersham ). Both antibodies gave identical results, detecting a single band of ∼70 kD that was present in Xenopus cytosol and (much less abundantly) in Xenopus membrane fractions. To visualize nuclear lamins by immunofluorescence, nuclei were assembled in the presence or absence of LAP2β fragments for 3 h. A 2.5-μl aliquot of each assembly reaction was placed on a slide and covered with the siliconized side of an 18 mm square coverslip. The slide was plunged into liquid N 2 for 10 s. Subsequent steps were performed at 22–24°C. The coverslip was quickly peeled off, and the sample was fixed/dehydrated in 100% methanol for 1 h. The slide was then rehydrated by incubation for 5 min each in 70, 50, and 30% methanol, then in PBS. After washing twice in PBS/0.1% Triton, and blocking for 5 min in PBS/0.1% Triton/2% BSA, the sample was incubated with 100 μg/ml mouse anti–human lamin B mAb ( Calbiochem-Novabiochem Corp. ) in PBS/0.1% Triton/2% BSA for 1 h. After extensive washes with PBS/2% BSA, the sample was incubated for 30 min with Texas red–conjugated goat anti–mouse antibody (Organon Teknika), washed twice with PBS/2% BSA, and incubated with the DNA dye Hoechst 33342 for 5 min. After three more washes with PBS/ 2% BSA, the sample was overlaid with 5 μl glycerol, covered with an 18 mm square coverslip, and viewed by phase-contrast and immunofluorescence on a Nikon Microphot fluorescent microscope. A Xenopus stage VI oocyte cDNA library in the UniZap vector (Stratagene Cloning Systems) was screened using full-length human LAP2β DNA as a probe. The probe was radiolabeled with α[ 32 P]dCTP using the Multiprime DNA labeling system (Nycomed Amersham ). The Xenopus cDNA library was a kind gift from D. Patterton and A. Wolffe (National Institutes of Health, Bethesda, MD). For the primary screen of 10 6 plaques, we used moderate stringency hybridization (30% formamide, 5× SSC, 42°C; washes in 2× SSC, 42°C), and obtained ∼300 positives. 20 strong positives were rescreened, and 12 remained positive. Single positive plaques from the tertiary screen were picked and converted to phagemids according to the manufacturer's protocol. Insert DNA was analyzed using restriction enzymes, revealing four distinct DNA inserts, which were sequenced. Sequences were aligned using ClustalW Multiple Sequence Align and BOXSHADE ( http://ulrec3.unil.ch/software/BOX_form.html ) software; DNA sequence information was further analyzed using DNA Strider. Clone 1 was a variant of clone 2, with several frameshifting point mutations, and was not studied further. GenBank accession numbers for Xenopus LAP2 clones are: clone 2 , clone 3 , and clone 4 . To construct Xenopus LAP2β fragment 1–164, a 5′ primer was designed to code for an Nde1 site followed by the first five amino acids 5′(GGGCATATGCCCGAGTTTCTG)3′, and a 3′ primer designed to encode the six terminal amino acids followed by an Xho1 site 5′(CCCCTCGAGTTCTTTATCACTGTAATG)3′. These primers were used in a PCR reaction to obtain a 510-bp fragment using clone 2 as template. This PCR product was digested with Nde1 and Xho1 (Life Technologies, Inc.; GIBCO BRL ) and ligated into corresponding sites in pET23a (Novagen, Inc.). In this case, the vector is predicted to express Xenopus LAP2β amino acids 1–164 followed by the six-His tag (173 amino acids; predicted mass of 19,749 D). The Xenopus fragment was expressed and purified as described above for the human LAP2 fragments. To explore LAP2β function, we purified two His-tagged recombinant polypeptides derived from human LAP2β . Fragment 1–408 (∼46.5 kD) included the entire nucleoplasmic portion of LAP2β, and is predicted to have both lamin-binding and chromatin-binding properties . Fragment 1–187 (∼21.7 kD) corresponds to the three conserved NH 2 -terminal exons present in all LAP2 isoforms and includes residues 1–85, which are sufficient for binding to chromatin . We used fragment 1–187 for two reasons. First, conserved exons frequently code for conserved structural domains within a protein, and second, fragment 1–187 was expected to compete with all endogenous LAP2 isoforms for binding to partners other than lamins. To determine its effects on nuclear assembly, fragment 1–408 was purified and added to Xenopus nuclear assembly reactions at concentrations ranging from 0.16 to 54 μM . Fragment 1–408 had no detectable effect on vesicle binding, envelope enclosure, or nuclear import (see below), but inhibited envelope growth at concentrations as low as 1–3 μM . The nuclei became enclosed by an intact nuclear envelope at the same time as control nuclei (∼30 min) but did not increase in size for at least 4.5 h. The final size of the arrested nuclei correlated inversely with the amount of fragment 1–408 in the reaction: at higher concentrations, the nuclei were smaller. At concentrations of 1.5–3 μM, fragment 1–408 reproducibly inhibited nuclear growth in all assembly extracts tested (three independent preparations), and virtually all nuclei were similar to those seen in Fig. 2 a. Inhibition was not due to residual imidizole, since control nuclei, which were assembled with proteins purified from uninduced bacteria, assembled and grew normally . Three lines of evidence showed that nuclei growth-arrested by fragment 1–408 were not defective for nuclear import. First, they contained prenucleolar coiled bodies , the formation of which is dependent on nuclear import . Second, they were active for DNA replication (see below), which also requires nuclear import. Third, we directly tested for import activity by first assembling nuclei for 2 h in the presence or absence of fragment 1–408, then adding a fluorescent karyophilic protein (rhodamine-conjugated nucleoplasmin), and imaging 30 min later. Nucleoplasmin accumulated at the nuclear rim and interior of the positive controls, and 1–408-arrested nuclei . Negative control nuclei, pretreated with the transport inhibitor WGA for 5 min before adding the nucleoplasmin, failed to accumulate the transport substrate . Because fragment 1–408 had no detectable effect on nuclear import in three independent assays, we concluded that the nuclear expansion defect was probably a direct effect of fragment 1–408 on other nuclear structures or pathways. Nuclei assembled for 3 h in the presence of purified fragment 1–187 appeared very different from 1–408-arrested nuclei, as seen by comparing Fig. 2 a (panel labeled 3.1 μM) with Fig. 3 a (upper right). Nuclei inhibited by fragment 1–187 remained small, and did not acquire a typical enclosed nuclear envelope. These effects were titratable over the low micromolar range. Nuclei assembled in the presence of 2–3 μM fragment 1–187 were smaller than positive controls. At 5–10 μM, the nuclear membranes consistently had an unusual scalloped morphology: some regions seemed flattened against the chromatin, whereas other regions appeared as oversized, unflattened vesicles. This scalloped appearance did not change for ≥5 h. The effects of fragment 1–187 were reproduced in three independent extracts. At a higher concentration (30 μM), fragment 1–187 delayed the attachment of membranes to chromatin : after 1–2 h of assembly, only a few patches of nuclear envelope were flattened onto chromatin. However, by 3 h the nuclear membranes had assembled enough to appear scalloped, like those assembled in lower concentrations (5–10 μM) of 1–187. These results showed that the putative chromatin-binding fragment of LAP2 inhibited membrane attachment to chromatin at a concentration of 30 μM. However, at lower concentrations the NH 2 -terminal fragment had quite different effects, interfering with both the enclosure and morphology of the nuclear envelope. To study their morphology in greater detail, nuclei were examined by transmission electron microscopy . As expected, control nuclei were enclosed by two nuclear membranes and studded with NPCs . Nuclei inhibited by fragment 1–408 had an enclosed nuclear envelope , confirming our phase-contrast observations . Although the particular cross-sections shown in Fig. 4 , a and b, are similar in size, the magnifications are different, and 1–408-inhibited nuclei were much smaller than control nuclei . TEM further revealed that 1–408-inhibited nuclei had numerous NPC-containing invaginations of the inner nuclear membrane . These invaginations were morphologically distinct from the nuclear tunnels described by Fricker et al. in which the entire envelope invaginates to form tubules extending into the nuclear interior. Nuclei inhibited by 1.5–3 μM fragment 1–408 also appeared to have a higher density of NPCs than control nuclei, consistent with ongoing NPC assembly into growth-arrested nuclei. TEM of nuclei assembled in the presence of 5 or 10 μM fragment 1–187 revealed that the chromatin was covered, but not enclosed, by nuclear membranes that contained NPCs . The envelope patches were concave relative to the chromatin, in contrast to the rounded convex shapes of control nuclei and nuclei inhibited by fragment 1–408 . We did not detect huge vesicles by TEM that might have corresponded to those seen by phase-contrast microscopy; we speculate that either these structures break during sample preparation, or that deeply concave sections of envelope might give the illusion of vesicles by light microscopy. The 1–187-arrested nuclei were similar to 1–408-arrested nuclei in two ways. First, they both had more NPCs than control nuclei, demonstrating that these LAP2 fragments do not interfere with NPC assembly. Second, they both had invaginations of the inner membrane into the nucleus, which might represent a problem in remodeling (or disconnecting) membrane-chromatin attachments (see Discussion). Our morphological results and import assays suggested that LAP2 proteins help shape the structural interface between the nuclear membranes and chromatin. We were particularly intrigued by the structural effects of fragment 1–187, which generated concave scallop-shaped envelopes. To our knowledge this phenotype is novel; for example, lamin-depleted nuclei are enclosed and round . To ask if LAP2 fragments affected lamina assembly, we assayed the inhibited nuclei for lamin accumulation . Nuclei were assembled for 3 h in reactions supplemented with either buffer, 3 μM fragment 1–408, or 5 μM fragment 1–187. Nuclei were then pelleted, subjected to SDS-PAGE, and immunoblotted with mAb 46F7, which is specific for lamin B3, the major lamin found in Xenopus eggs . Identical results were obtained using an mAb raised against human lamin B (data not shown). Nuclei inhibited by fragment 1–408, but not those inhibited by fragment 1–187, accumulated lamins over time as shown by immunoblotting of pelleted nuclei . The lamin signal in 1–408-inhibited nuclei was sometimes slightly stronger than in control nuclei (two of five experiments), perhaps reflecting the presence of additional lamin-binding proteins (i.e., fragment 1–408) in these nuclei. These lamin results were consistent with the structural results, since 1–408-inhibited nuclei were enclosed and active for nuclear protein import, and 1–187-inhibited nuclei were not enclosed and did not accumulate imported proteins such as lamins. We also used indirect immunofluorescence to detect lamins in inhibited nuclei . Consistent with the lamin blots, lamins were not detected by indirect immunofluorescence in 1–187-inhibited nuclei. Lamins were detected both in control nuclei and nuclei inhibited by fragment 1–408, in close association with the nuclear envelope . However, because the 1–408-inhibited nuclei were small, and the immunofluorescent signal quite bright , we could not determine unambiguously whether lamins were associated exclusively with the envelope, or if they might have also accumulated at inappropriate sites inside the nucleus. As a negative control for the DNA replication experiments described below, we used GTPγS to inhibit vesicle fusion . No replication was observed in GTPγS-treated reactions, as expected, since these nuclei consist of small vesicles bound to the chromatin surface. However, the GTPγS-arrested nuclei accumulated a low level of lamins , suggesting that some interactions involving lamins may have proceeded to a limited extent even though vesicle fusion was inhibited by GTPγS. The modest accumulation of lamins on GTPγS-arrested nuclei contrasted significantly with 1–187-inhibited nuclei, which had only background amounts of lamins . We concluded that fragment 1–187 blocks lamin recruitment or attachment to the membrane–chromatin interface. Because there is strong evidence that the NH 2 -terminal region of LAP2 binds to chromatin , we further concluded that fragment 1–187 binds competitively to, and blocks, the chromatin partners for endogenous LAP2 proteins. These findings therefore suggest that endogenous LAP2 isoforms must engage their chromatin partner as a prerequisite for lamina assembly. DNA replication normally occurs in in vitro assembled nuclei if the lamina is properly assembled . Therefore, replication is used as a marker for the assembly of a structurally intact nucleus. Since nuclei inhibited by fragment 1–187 did not accumulate lamins, we predicted that these nuclei would be unable to replicate. We did not know what to expect with 1–408-inhibited nuclei, which accumulated lamins. Fragment 1–408 is predicted to bind lamins , and might somehow disrupt lamina assembly, and hence, secondarily disrupt replication. To test DNA replication, nuclei were assembled in the presence of [ 32 P]dCTP for 3 h, with or without added LAP2 fragments, and the DNA was analyzed by agarose gel electrophoresis and autoradiography . Aliquots were processed in parallel for Western blotting with an anti-lamin antibody , and a representative nucleus from each sample is shown by phase-contrast . No replication activity was detected in 1–187-arrested nuclei , as predicted from their lack of enclosure. However, nuclei assembled in the presence of 3 μM fragment 1–408 consistently replicated at levels as high (three experiments) or higher (eight experiments) than control nuclei . In the experiment shown, 1–408-inhibited nuclei also accumulated lamins at higher levels than controls . The increased replication signal in 1–408-inhibited nuclei was not due to unequal loading . No replication was detected in negative controls treated with GTPγS to prevent envelope formation , or in the presence of 50 μg/ml aphidicolin , a specific inhibitor of DNA polymerase α . Note the modest degree of enhancement by fragment 1–408 in Fig. 7 , an example of the low end of experimental variation in the extent of enhancement. We concluded that although the LAP2 fragment 1–408 blocked the expansion of nascent nuclei in vitro, it unexpectedly enhanced DNA replication activity. Based on densitometry quantitation of eight experiments, nuclei arrested by fragment 1–408 replicated an average of 2.5-fold better than controls. We considered two different mechanisms for the increase in [ 32 P]dCTP incorporation: fragment 1–408 might cause rereplication, which would represent a loss of cell cycle control, or it might enhance semiconservative DNA replication, the efficiency of which can vary from 30 to 100% in Xenopus egg extracts . The first possibility, rereplication, was tested by equilibrium density substitution in the presence of a dense nucleotide derivative, BrdU . These reactions also contained [ 32 P]dCTP, which allowed us to detect replicated strands, and quantitate the extent of replication (see Materials and Methods). The positive control (no LAP2 fragment) was expected to undergo a single round of semiconservative replication to yield DNA with one light strand and one heavy strand, which would migrate as a single peak in a CsCl gradient. If the enhanced replication were due to rereplication, we would detect a second peak of heavy–heavy DNA at a higher density, and we would not expect the heavy–light DNA from LAP2-treated nuclei to have significantly more radioactivity than the positive control. The experiment was done using both fractionated/reconstituted extract (data not shown) and fresh 10,000 g crude cytoplasm . We found that LAP2-arrested nuclei yielded a single peak of radiolabeled DNA that comigrated at exactly the same refractive index, and hence, density, as the positive control, indicating a single round of semiconservative DNA replication. Furthermore, the peak of incorporated nucleotide was at least fourfold higher than the positive control, consistent with fragment 1–408 enhancing the efficiency of semiconservative replication. These results effectively ruled out the possibility that LAP2 fragment 1–408 causes rereplication. To examine the time course of replication in LAP2-arrested nuclei, nuclei were assembled in reactions containing α[ 32 P]dCTP in the presence or absence of fragment 1–408; aliquots were removed from each reaction every 15 min, and the incorporated radiolabel was quantitated . The time course of replication was initially indistinguishable between LAP2-arrested nuclei and positive controls: there was a lag phase of 45 min, followed by DNA synthesis. The only difference was that α[ 32 P]dCTP incorporation into LAP2-arrested nuclei continued at the same rate for ≥15 min longer than positive controls, reaching a plateau that was, in this case, ∼1.8-fold higher than the positive control . The replication results collectively led us to two conclusions. First, the majority of our extracts, which were made from unactivated eggs, were not 100% efficient for replication. Based on the amount of replication enhancement seen in these experiments, which ranged from 0 to ∼4-fold (average, 2.5-fold), we estimated that the efficiency of replication in our extracts varied from 20% to 100%. Second, in extracts that were <100% efficient for replication, LAP2 fragment 1–408 increased the efficiency of semiconservative DNA replication, when present at low (2–4 μM) concentrations. Possible mechanisms for the enhancement of replication efficiency by LAP2 are considered in the Discussion. Given the effects of human LAP2 fragments in Xenopus nuclear assembly extracts, it was essential to determine if Xenopus LAP2 had the same structural effect on nuclear assembly. To do this, and compare the Xenopus and human LAP2 proteins, we screened a stage VI Xenopus oocyte cDNA library using radiolabeled full-length human LAP2β as a probe (see Materials and Methods). We rescreened 20 of nearly 300 positives, and identified three cDNAs coding for Xenopus LAP2β homologues, which were designated clones 2, 3, and 4 . The three Xenopus LAP2 proteins are compared schematically to human LAP2β in Fig. 9 a. Clone 2 was the longest Xenopus LAP2 cDNA, encoding a protein of predicted mass 62.841 kD. Except for a single-base deletion at nucleotide 1119 in clone 3, which is either a mutation or the result of an alternative splicing event (see below), the three Xenopus cDNAs were identical at the nucleotide level except for two regions: nucleotides 595–705 and 1068–1278 in clone 2. These two regions encoded polypeptide inserts that we named insert A (37 residues, 198–234), insert B (17 residues, 357–373), and insert C (53 residues, 374–426), as diagrammed in Fig. 9 a. Clone 2 had all three inserts, whereas clone 3 lacked insert C, and clone 4 lacked insert A, suggesting that inserts A and C were alternatively spliced exons. Although insert B was present in all three Xenopus cDNAs, it was absent from human LAP2 and is therefore either a new exon, or a nonhomologous extension of the neighboring exon. All three putative new exons were located at exon boundaries in the mouse genomic sequence : insert A between mouse exons 5 and 6, and inserts B and C between mouse exons 8 and 9. We concluded that inserts A and C (and probably B) represent bona fide LAP2 exons in Xenopus . Exons homologous to inserts A, B, and C have not yet been reported in mammals. Overall, these results suggested that Xenopus clones 2, 3, and 4 were splicing variants related to mammalian LAP2β. Notably, clone 3 encoded a putative β-related isoform that lacks a transmembrane domain, similar to the ζ (zeta) isoform in mice . Three regions were broadly similar between Xenopus and human LAP2β proteins: an NH 2 -terminal region , a middle region (diagonal bars), and a COOH-terminal region (horizontal stripes). The protein encoded by clone 2 is compared with human LAP2β in detail in Fig. 9 b. The NH 2 -terminal region of clone 2 (residues 1–197) was 70% identical and 80% similar to the same region in human LAP2 (residues 1–220). The middle region was less conserved: residues 234–352 of clone 2 were 39% identical and 53% similar to human LAP2β residues 220–325. At the COOH-terminal region, Xenopus residues 427–556 were 63% identical and 74% similar to human LAP2β residues 330–453. We concluded that the middle region of LAP2β is the least conserved between species, in contrast to the more-conserved NH 2 -terminal and COOH-terminal domains. To assess the effects of Xenopus LAP2 on nuclear assembly, we focussed on the NH 2 -terminal domain. We expressed and purified the Xenopus LAP2 fragment consisting of residues 1–164, which are homologous to human fragment 1–187, and added it to Xenopus nuclear assembly reactions at concentrations ranging from 1 to 10 μM . The Xenopus LAP2 fragment had structural effects identical to those of human LAP2 fragment 1–187, producing scalloped, nonenclosed envelopes at concentrations of 1, 2.5, and 5 μM . At 10 μM, fragment 1–164 interfered with membrane targeting to the chromatin surface , paralleling the effects of human fragment 1–187 at 30 μM . We noted that Xenopus fragment 1–164 was inhibitory at concentrations two to five times lower than the comparable human fragment. We concluded that the human and Xenopus NH 2 -terminal fragments of LAP2 had the same effects on nuclear assembly in vitro. These results strongly suggested that the human polypeptides interacted with bona fide LAP2 binding partners in Xenopus extracts, with slightly lower efficiency, presumably due to species-specific differences in amino acid sequence. We concluded that the human LAP2 fragments used in our experiments affected nuclear assembly and DNA replication by competing for the binding partners of endogenous LAP2 proteins. Consistent with previous results from microinjected HeLa cells , our in vitro results show that a LAP2β fragment capable of binding to lamins has the effect of blocking nuclear expansion after enclosure. This finding confirms the importance of LAP2β and lamins in mediating nuclear growth. We further show that nuclei growth-arrested by the chromatin-and-lamin-binding nucleoplasmic domain of LAP2β (residues 1–408) were enhanced in their efficiency of semi-conservative DNA replication; this result has important new implications for LAP2 function, as discussed below. In contrast to Yang et al. , who found that residues 1–85 of the conserved NH 2 -terminal domain had no effect in vivo, the complete NH 2 -terminal chromatin-binding domain of both human and Xenopus LAP2 strongly inhibited nuclear assembly, producing a scalloped envelope morphology and blocking lamina assembly. The implications of this phenotype are discussed below. Several studies suggest that the LAP2 isoforms are collectively responsible for dynamically organizing the lamina: the biochemical demonstration that rat LAP2β binds lamin B , the sequential colocalization of LAP2α with lamin B and lamin A during nuclear assembly in vivo , the in vivo and in vitro nuclear growth arrest by lamin-binding fragments of LAP2β , and the block to lamin accumulation caused by the NH 2 -terminal chromatin-binding domain of LAP2 (human residues 1–187; this study). The finding, that nuclei arrested by fragment 1–187 did not accumulate lamins, was unexpected since this shared domain of LAP2 binds to chromatin, not lamins. We suggest that when the recombinant chromatin-binding domain of LAP2 is added to assembly reactions, it occupies chromatin sites and prevents endogenous LAP2 proteins from attaching to chromatin. To explain how fragment 1–187 then prevents lamin assembly, we propose that the endogenous LAP2 proteins need to bind to chromatin as a prerequisite for binding to lamins and promoting lamin assembly. We characterized three cDNAs from Xenopus , which appear to be new β-related isoforms of LAP2. These cDNAs include three putative novel exons, referred to as inserts A, B, and C. Insert A is positioned immediately after the conserved NH 2 -terminal domain. Inserts B and C interrupt the minimal lamin-binding region of LAP2β . Interestingly, the minimal lamin-binding region is not encoded by a single exon, but spans exons 8 and 9 . Xenopus inserts B and C are located precisely between mice exons 8 and 9, inserting 17 and 53 residues, respectively, into the middle of the lamin-binding region. Lamin-binding activity is influenced by the COOH-terminal region of LAP2, since residues 298–452 of rat LAP2β bind to lamins fivefold better than minimal residues 298–373 in a yeast two-hybrid assay . In view of our hypothesis that LAP2 chromatin-binding activity may regulate or promote lamin recruitment, it will be interesting to determine the lamin-binding and other activities of Xenopus LAP2 isoforms that have, or lack, each new exon. One explanation for why fragment 1–408 (the chromatin-and-lamin-binding fragment) arrests the expansion of nascent nuclei is that it may contribute to the formation of excess or unregulated connections between lamins, membranes, and chromatin. This possibility is supported by the extensive invagination of the inner membrane seen in nuclei arrested by fragment 1–408. However an alternative possibility, that this chromatin-and-lamin-binding LAP2 fragment inhibits chromatin decondensation, is supported by our finding that high concentrations (54 μM) of fragment 1–408 caused the sperm chromatin to remain smaller than the size expected of chromatin swelled by exposure to egg cytosol . The association of LAP2 with chromatin is relatively strong, since immunoaffinity-purified LAP2β binds saturably to mitotic chromosomes with an affinity of 40–80 nM . We hypothesize that at high concentrations, recombinant LAP2β may either block chromatin decondensation, or promote condensation. Interestingly, a chromatin binding partner for LAP2 has been provisionally identified by two-hybrid analysis in yeast as BAF . BAF localizes to the nucleus during interphase, and to chromosomes during mitosis . BAF is a small novel cellular protein (89 residues) that was identified because it facilitates the efficient integration of HIV DNA into the cell's genome ; in the absence of BAF, the viral DNA molecule tends to integrate intramolecularly into itself. Lee and Craigie propose that BAF acts by crossbridging and thereby compacting the viral DNA molecule. The normal cellular role of BAF is not known. If further experiments confirm that BAF and LAP2 are indeed binding partners, it will be interesting to test the idea that LAP2 influences chromatin structure by affecting BAF activity. The minimal lamin-binding region of LAP2β (rat residues 298–373) inhibits the initiation, but not the progression, of DNA replication in vivo . Consistent with this, the full nucleoplasmic region of human LAP2β (fragment 1–408) also inhibited DNA replication at concentrations above 6 μM (data not shown). However at lower concentrations (1–3 μM), which reproducibly inhibited nuclear expansion, fragment 1–408 enhanced DNA replication activity by an average of 2.5-fold in ∼80% of our experiments. Thus, by varying the concentration of recombinant protein in the reaction, we uncovered a positive role for LAP2β in replication. Based on density-substitution experiments, we eliminated the possibility that increased nucleotide incorporation was due to rereplication. A second possibility, that fragment 1–408 triggers extreme levels of repair synthesis, seems unlikely, but cannot be ruled out by our present data. Our results favor a third possibility, that LAP2β fragment 1–408 increases the efficiency of semiconservative DNA replication. This increased efficiency could be an indirect effect of LAP2 on nuclear size, since the small arrested nuclei may achieve higher concentrations of imported replication factors. For example, chromatin can replicate efficiently in the absence of nuclei in vitro, by sequential exposure to cytosolic extracts and 25-fold concentrated nucleoplasmic extracts . We can provisionally rule out such a size based model for one simple reason: 1–408-arrested nuclei were always small, but replication was not enhanced in ∼20% of our experiments (see below). We think this extract-to-extract variation is an important clue about the mechanism of enhancement. Logically, replication enhancement is only possible in extracts that are less than 100% efficient. Xenopus egg extracts can vary in replication efficiency from 30% to 100% . The cause of this variation is not yet known, but we hypothesize that it may reflect differences between extracts in the efficiency with which prereplication complexes assemble onto chromatin. The prereplication complex consists of MCM proteins, the cdc6 protein, and six ORC proteins; this complex can only be assembled when there are no cyclin-dependent kinases (CDKs) active in the cell . In somatic cells, such a situation only exists for a narrow window of time between anaphase (when the mitotic cyclin-dependent kinases are inactivated) and early G1 (when G1-phase kinases are activated). According to the two-step model for replication control, which is widely supported by evidence from yeast and Xenopus , the prereplication complex is the obligatory precursor of the replication complex, and is removed during replication, thereby providing a mechanism to limit replication to a single round per cell cycle . Eggs have high levels of mitotic CDK activity (also known as maturation promoting factor activity), which maintains them in a metaphase-arrested state until fertilization. Fertilization triggers a wave of intracellular Ca 2+ release, which destroys the Ca 2+ -sensitive cytostatic factor stabilizing maturation promoting factor . Many investigators mimic fertilization by either exposing eggs to Ca 2+ or electric shock, before making extracts. In contrast, our extracts are from unactivated eggs; activation is not essential to obtain extracts competent for interphase nuclear assembly , probably because unactivated eggs are exposed to contaminating Ca 2+ ion during extract preparation. To explain our extract-to-extract variation in both replication enhancement per se (no enhancement in ∼20% of experiments) and the degree of enhancement (up to fourfold; averaging 2.5-fold), we hypothesize that in many of our extracts the mitotic CDK activity is not fully inactivated. Trace CDK activity might decrease the number of prereplication complexes that can assemble, and thus reduce the efficiency of replication. Further experiments are needed to test this hypothesis. Nuclei assembled in lamin-depleted extracts cannot undergo DNA replication , initially suggesting that lamina assembly is required for DNA replication initiation or progression. However as noted above, replication can occur in the absence of nuclei if the chromatin is exposed to concentrated nucleosolic extracts . The replication-promoting components of these nucleosolic extracts have not yet been identified, and are likely to include multiple factors potentially including LAP2 isoform(s). Based on our evidence that LAP2 may affect chromatin structure, and that the full nucleoplasmic portion of LAP2β (fragment 1–408) enhances the efficiency of DNA replication, we hypothesize that LAP2 isoform(s) may act as downstream effectors of lamina assembly by promoting chromatin conformations favorable either to the assembly of prereplication complexes, or the progression of replication complexes. Further experiments are needed to determine if the putative chromatin-influencing activities of LAP2 proteins depend on lamin assembly, if they regulate lamin assembly, or both. The idea that LAP2β might promote replication by affecting chromatin structure has a precedent when one considers LBR, the lamin B receptor. LBR interacts with a chromatin partner named Hp1 , which mediates repressive higher-order chromatin structure in Drosophila . In turn, Drosophila Hp1 is known to bind ORC1 . Thus, LBR (an inner nuclear membrane protein) binds to Hp1, which can bind a core component of the prereplication complex. The meanings and mechanisms of these interactions are not yet clear. However, it appears that LBR, and perhaps LAP2, may influence chromatin structure in ways that could modulate the competence of chromatin for DNA replication, and also conceivably its competence for transcription. Our identification of new exons in the lamin-binding region of Xenopus LAP2 isoforms increases the potential for subtle differences in functions of the various LAP2 isoforms. Further analysis of the effects of LAP2 isoforms on nuclear dynamics, chromatin structure, DNA replication, and potentially the transcriptional competence of chromosomes will be of interest. | Study | biomedical | en | 0.999996 |
10087256 | Rat liver nuclei were isolated as described and stored frozen at −80°C in 100 unit aliquots (1 U = 3 × 10 6 nuclei). Nuclear envelopes were prepared by a modification of the procedure described by Dwyer and Blobel . Nuclei were thawed and pelleted at 500 rpm in a tabletop microfuge for 1 min. After removing the supernatant, the pellet was resuspended to a final concentration of 100 U/ml by drop-wise addition of cold buffer A (0.1 mM MgCl 2 , protease inhibitors [0.5 mM phenylmethanesulfonyl fluoride {PMSF}, 1 μg/ml leupeptin, 1 μg/ ml pepstatin A, and 18 μg/ml aprotinin], 5 μg/ml DNase I [ Sigma Chemical Co. ], and 5 μg/ml RNase A [ Sigma Chemical Co. ]) with constant vortexing. The nuclei were then immediately diluted to 20 U/ml by addition of ice-cold buffer B (10% sucrose, 20 mM triethanolamine (pH 8.5), 0.1 mM MgCl 2 , 1 mM DTT and protease inhibitors), again with constant vortexing. The suspension was dounced four times in a glass dounce homogenizer (tight pestle) and incubated at room temperature for 15 min. After the 15-min incubation, the suspension was underlaid with 5 ml of ice-cold buffer C (30% sucrose, 20 mM triethanolamine, pH 7.5, 0.1 mM MgCl 2 , 1 mM DTT and protease inhibitors) and centrifuged at 4,100 g in a swinging bucket rotor (Sorvall type HB-4) for 15 min at 4°C. After removing the supernatant and sucrose cushion, the pellet was resuspended to a final concentration of 100 U/ml in ice-cold buffer D (10% sucrose, 20 mM triethanolamine, pH 7.5, 0.1 mM MgCl 2 , 1 mM DTT and protease inhibitors). The suspension was dounced as above, and diluted to 66 U/ml by addition of cold buffer D containing 0.3 mg/ml heparin ( Sigma Chemical Co. ). The suspension was immediately underlaid with 5 ml of buffer C and pelleted as above. The pellet resulting from this second extraction is operationally defined as the nuclear envelope fraction. After removing the supernatant and sucrose cushion, the pellet was resuspended to a final concentration of 100 U/ml in ice-cold buffer D. The suspension was dounced as above and diluted to 66 U/ml by addition of cold buffer D containing 3% Triton X-100 and 0.075% SDS. The suspension was immediately underlaid with 5 ml of buffer C and pelleted as above. The pellet resulting from this second extraction is operationally defined as the pore complex lamina fraction (PCLF). The PCLF was resuspended in cold buffer D at 100 U/ml, dounced as above, and diluted to 66 U/ml with buffer D containing 0.9% Empigen BB ( Calbiochem-Novabiochem ). Samples were incubated on ice for 5 min and subsequently pelleted for 15 min at 15,000 g for 15 min. The supernatant fraction, containing nucleoporins minus the nuclear lamins, was precipitated with 10% TCA. Precipitated proteins were resuspended in sample buffer, and the equivalent of 10 U of nuclei were analyzed by Coomassie blue staining. The PCLF was isolated as described above and resuspended (1,000 U/ml) in buffer D plus 1% Triton X-100, 0.025% SDS, and 0.1 mg/ml heparin. After resuspension, the samples were immediately spun at 15,000 g for 10 min at 4°C. The supernatant was then loaded onto a 10–40% sucrose gradient (triethanolamine pH 7.5, 0.1 mM MgCl 2 ) and spun at 40,000 rpm in a Beckman SW 41 rotor for 17 h at 4°C. 750-μl fractions were collected, TCA precipitated and analyzed by SDS-PAGE and subsequent silver staining, or immunoblotting. All clones and constructs were sequenced by the Rockefeller University Protein/DNA Technology Center using an ABI automated DNA sequencer. To isolate cDNAs encoding for Nup96, a 48-nucleotide (nt) ssDNA probe was synthesized based on the region of clone GM2B8 (kindly provided by Dr. Mark Rutherford, University of Minnesota, St. Paul) coding for the sequenced peptide “LVVRHLASDAIINENYD” . This oligonucleotide probe was used to screen a HeLa cDNA library (kindly provided by Dr. Jian Wu, Rockefeller University, New York). Two classes of cDNA clones, differing by a 225-nt insert, were obtained in approximately equal numbers. All of the isolated clones had incomplete 5′ ends, and 5′ RACE was therefore performed using human fetal liver cDNA ( Clontech Laboratories ) as template. The 5′ RACE product and the longest clone obtained from the HeLa cDNA library were assembled at an overlapping ACC I site, and the full-length clone was then ligated into the SalI and NotI sites of a modified pAlter-MAX vector ( Promega Corp. ) containing an in-frame myc epitope tag. Modified myc-pAlter-MAX, containing the myc epitope tag, has been previously described . Myc-tagged Nup96 was generated by PCR using the wild-type Nup98-Nup96 cDNA as template. An oligonucleotide complementary to the 5′ end of the Nup96 ORF (beginning with serine 864) and containing a SalI site was used in conjunction with an antisense oligonucleotide complementary to sequences at the 3′ end of the Nup98-Nup96 ORF and containing a NotI site. The resulting PCR product was digested with SalI and NotI and ligated into the SalI/NotI sites of myc-pAlter-MAX. The myc-tagged, full-length Nup98 was generated using a similar PCR based approach using the Nup98-Nup96 cDNA as template. An oligonucleotide complementary to the 5′ end of the Nup98-Nup96 ORF was used in conjunction with an antisense oligonucleotide complementary to the 3′ end of the Nup98 ORF (ending with proline 912). The oligonucleotide also contained an additional 18 nt coding for the 6 COOH-terminal amino acids of full-length human Nup98 that are not encoded by the Nup98-Nup96 precursor site and a NotI restriction site. The resulting PCR product was digested with SalI and NotI and ligated into the SalI/NotI sites of myc-pAlter-MAX. The cleaved form of Nup98 (amino acids 1–863) was also generated by PCR using the wild-type Nup98-Nup96 cDNA as template, and the resulting PCR product was cloned into the SalI/NotI sites of myc-pAlter-MAX. The Nup98-Nup96 precursor double mutant (F863S/Y866R), and the similar Nup98 double mutant, were generated by recombinant PCR using mismatched primers and the myc-tagged Nup98-Nup96 precursor cDNA as template . Proteins from fractionated nuclear envelope were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The appropriate proteins were excised after identification by staining with Ponceau S. The proteins were extracted or were digested on the membrane with endoproteinase Lys-C. Peptides were separated and sequenced as previously described . For protein production, a DNA fragment corresponding to amino acids 1291–1482 of the Nup98-Nup96 precursor was obtained by PCR and cloned into the NdeI and AvaI sites of the bacterial expression vector pET21a (Novagen, Inc.). E. coli strain BL21 (DE3) pLysE was transformed with the expression vector, and expression of the recombinant protein was induced by adding 0.2 mM isopropyl thio-β- d -galactoside to the culture media. The recombinant protein was purified by Nickel-NTA agarose as described by the manufacturer (QIAGEN Inc.). For antibody production, rabbits were injected with purified, recombinant protein and serum was collected after an appropriate response had been elicited. Antibodies were purified from the crude serum by affinity chromatography. The recombinant protein was immobilized on Affi-Gel 15 according to the manufacturer (Bio-Rad Laboratories), and serum was incubated with the gel for 12 h. The gel was loaded into a column, and the column was washed with 1 M NaCl in PBS. Bound antibodies were eluted with 0.1 M glycine (pH 2.5), and the eluate immediately neutralized with 1 M Tris (pH 8.0). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane as previously described . Nitrocellulose membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% NP-40, and the blots were probed with affinity-purified Nup96 antibodies diluted 1:300 in PBS containing 2% BSA. Nup107 was detected using an affinity-purified rabbit polyclonal antibody . Antibodies were detected using luminol-based chemiluminescence. HeLa cells were grown in DME supplemented with 10% fetal calf serum. For immunofluorescence, cells were grown overnight on coverslips, washed in PBS, fixed in 2% formaldehyde/PBS for 30 min at room temperature, and permeabilized with −20°C acetone for 3 min. Affinity-purified Nup96 antibodies (diluted 1:300), the anti-myc monoclonal antibody 9E10 , and rabbit anti-Nup358 antibodies were incubated with the cells for 1 h at room temperature. Cells were washed in PBS and incubated with either fluorescein-conjugated goat anti–rabbit (Organon/Teknika), and/or Cy3-conjugated donkey anti–mouse (Jackson ImmunoResearch) antibodies for 30 min at room temperature. Cells were washed again in PBS and mounted in 80% glycerol, 50 mM Tris-HCl (pH 8.0), 0.1% p -phenylenediamine. Samples were analyzed with a Zeiss laser scanning confocal microscope. For transfections, HeLa cells were grown on 35-mm dishes and were transfected with 2 μg of DNA using lipofectamine ( GIBCO-BRL ) according to the manufacturer's instructions. After 36 h, cells were assayed either by immunofluorescence microscopy or by immunoblot analysis as described above. Rat liver nuclear envelopes were prepared as described above. Isolated nuclear envelopes were fixed for 15 min in 2.5% formaldehyde in STM (10% sucrose, 20 mM triethanolamine-HCl, pH 7.5, 0.1 mM MgCl 2 ) and centrifuged at 2,000 g for 5 min onto 35-mm tissue culture dishes. The pelleted nuclear envelopes were washed three times with 1% BSA, 68 mM NaCl, 13 mM KCl, 15 mM KH 2 PO 4 , 40 mM Na 2 HPO 4 , and 0.5 mM PMSF and then incubated with affinity-purified anti-Nup96 antibodies diluted 1:300, and mouse monoclonal antibody 19C7 , or mouse monoclonal antibody 5E10 (specific for Tpr; Matunis, M.J., and G. Blobel, unpublished data) diluted 1:1,000. Bound antibodies were detected with goat anti–rabbit IgG conjugated with 10-nm gold, or goat anti–mouse IgG conjugated with 5-nm gold ( Amersham Life Science Inc.). For in situ labeling of intact cells, HeLa cells grown on 35-mm tissue culture dishes were first permeabilized with digitonin (50 μg/ml) for 5 min at room temperature, fixed with 2% formaldehyde in PBS for 30 min at room temperature, incubated with primary and secondary antibodies as indicated above and processed for thin sectioning and EM as previously described . All Nup98 and Nup98-Nup96 precursor proteins were synthesized in rabbit reticulocyte lysate transcription and translation extracts, in the presence of [ 35 S]methionine, as described by the manufacturer ( Promega Corp. ). Proteins were separated by SDS-PAGE and analyzed by autoradiography. We have developed a novel procedure for purifying the protein components of the mammalian NPC . In the final step of this procedure, NPCs are solubilized and released from the nuclear lamina by extraction with the zwitterionic detergent, Empigen BB. The Empigen BB supernatant fraction contains all of the currently known nucleoporins, in addition to ∼30 uncharacterized, candidate nucleoporins . One of the major uncharacterized proteins, with an apparent molecular mass of 115 kD , was analyzed by peptide sequence analysis, and the peptide sequence was used to isolate a full-length cDNA clone encoding this protein (see following sections). Based on its predicted molecular mass of 96 kD, and its association with NPCs, this novel nucleoporin has been termed Nup96. Nup96 runs anomalously at 115 kD by SDS-PAGE. To produce antibodies specific for Nup96, a 22-kD fragment was expressed in E. coli and injected into rabbits. Affinity-purified antibodies reacted with Nup96, as indicated by immunoblot analysis of SDS-PAGE separated proteins of total nuclei and isolated nuclear envelopes . Fractionation of isolated rat liver nuclei showed that Nup96 is associated predominantly with the nuclear envelope , although a significant fraction was also detected in the nucleoplasmic fraction , consistent with the immunofluorescence data (see below). In addition to Nup96, the affinity-purified antibodies also recognized a 200-kD protein that fractionated exclusively with the soluble nucleoplasm . As Nup96 is synthesized as a 186-kD protein that is proteolytically cleaved in vivo (see following sections), this 200-kD protein could represent the intact precursor. However, a similar band is not detected with antibodies to Nup98 (data not shown), and pulse-labeling experiments indicate that the precursor has a very short half-life (data not shown). Therefore, it is more likely that this band corresponds to a cross-reacting protein unrelated to the Nup98-Nup96 precursor. By indirect immunofluorescence microscopy Nup96 showed a rim-like staining at the midplane of the nucleus . The nuclear envelope staining became more diffuse and punctate upon focusing near the surface of the nucleus (data not shown). This labeling pattern is characteristic of that observed for other nucleoporins , and is further evidence for the association of Nup96 with NPCs. In addition to nuclear envelope staining, a weak intranuclear signal was also observed. This signal may be attributed to the presence of Nup96 within the nucleus, and/or to the 200-kD protein with which the Nup96 antibodies cross-react . Signal was excluded from the nucleoli. To further sub-localize Nup96 at the level of the NPC, immunogold EM was performed using anti-Nup96 antibodies. When isolated rat liver nuclear envelopes were probed, Nup96 was detected specifically at the nucleoplasmic face of the NPCs . Gold particles were distributed over a range from 22 to 50 nm from the mid-plane of the nuclear envelope, with a mean distance of 36 nm ( n = 50). The relative position of Nup96 in the NPC was established by double labeling experiments using monoclonal antibodies to RanGAP1 and Tpr , markers for cytoplasmic and nucleoplasmic fibers of the NPC, respectively . Nup96 was located at or near the nucleoplasmic basket of the NPC, on the opposite face compared with RanGAP1, and in close proximity to Tpr. Because components of the NPC fibers may be lost or the fibers may collapse during isolation of nuclear envelopes, we also examined the localization of Nup96 in intact cells. Cells were permeabilized using digitonin, fixed and labeled with both primary and secondary antibodies before processing and sectioning for EM. Under these conditions, the relative distributions of Nup96 , RanGAP1 , and Tpr remained the same, confirming the results obtained using isolated nuclear envelopes. However, the distance of Nup96 from the mid-plane of the nuclear envelope was dramatically greater in the intact cells (averaging 100–200 nm), compared with the distance observed with isolated nuclear envelopes (averaging 36 nm). This difference could be due to the collapse of the nucleoplasmic basket and/or attached filaments upon isolation of nuclear envelopes. The internal peptide sequence LVVRHLASDAIINENYD derived from Nup96 was used to search sequence databases for candidate cDNA clones coding for Nup96. A partial cDNA clone was identified, GM2B8, that contained sequences coding for this peptide. GM2B8 was originally derived from a mouse macrophage subtraction library designed to identify messenger RNAs induced by interferon-γ . Screening a HeLa cell cDNA library with a GM2B8 probe and subsequent 5′ RACE yielded full-length clones coding for Nup96 (see Materials and Methods). Surprisingly, the longest full-length clone obtained predicted a protein of 1,712 amino acids, with a molecular mass of 186 kD . This size was in contrast to the expected 115 kD determined by SDS-PAGE . Analysis of the predicted amino acid sequence of this clone revealed that the NH 2 -terminal 914 residues are identical to the previously characterized nucleoporin, Nup98 , whereas the remaining 798 COOH-terminal residues are unique and contain the sequenced peptide derived from Nup96. Because this predicted protein contains sequences identical to both Nup98 and Nup96, it will be referred to as the Nup98-Nup96 precursor. Previously identified, complete cDNA clones for Nup98 predict a protein product of 920 amino acids . The Nup98-Nup96 mRNA does not contain the last 18 nucleotides coding for the COOH-terminal 6 amino acids of Nup98. Several lines of evidence suggest that the mRNA coding for the Nup98-Nup96 precursor, and the previously described Nup98 mRNA, are derived from a common, alternatively spliced pre-mRNA. First, the nucleotide sequences in both the 5′ untranslated regions and in the Nup98 coding regions are 100% identical between both mRNAs . Second, Northern blot analysis with a Nup98-specific probe revealed multiple RNA transcripts of ∼4, 6, 6.5, and 7 kb . A Nup96-specific probe detected similar 6-, 6.5-, and 7-kb transcripts, but not a 4-kb transcript . These data suggest that the larger transcripts code for the Nup98-Nup96 precursor, whereas the shorter 4-kb transcript codes independently for Nup98. With regard to the three Nup98-Nup96 precursor transcripts detected by Northern blot analysis, two classes of cDNAs were isolated during the screen for Nup96 clones. These cDNAs encode identical proteins, with the exception of a 75–amino acid insert predicted by the longer clone . Because the protein containing this 75– amino acid insert comigrates with endogenous Nup96 , whereas the protein without this insert does not (data not shown), the longer clone was used for subsequent analysis. When the entire Nup98-Nup96 precursor sequence was aligned to all previously known proteins, significant homology was observed with the S. cerevisiae nucleoporin, Nup145p, as well as with an ORF located on chromosome I of Arabidopsis thaliana , and a protein predicted by the Caenorhabditis elegans cDNA, ZK328.5b . The NH 2 -terminal domains of the Nup98-96 precursor, Nup145p, and the C. elegans protein, all predict polypeptides with conserved GLFG repeats. The A. thaliana protein does not contain a GLFG-repeat domain. Near the middle of the Nup98-Nup96 precursor, Nup145p, and the C. elegans protein, and near the NH 2 terminus of the A. thaliana protein, is a highly conserved domain . This domain is of particular interest, because in S. cerevisiae , Nup145p is posttranslationally cleaved at a site near the COOH-terminus of the domain (after the phenylalanine at position 605), thereby generating two independent nucleoporins, N-Nup145p and C-Nup145p . The high conservation of the cleavage site and surrounding residues between yeast, plant, worm, and mammal suggested that the posttranslational processing pathway may be conserved and functionally important. After the highly conserved cleavage site, all four proteins continue to be homologous over their entire length . The COOH-terminal domains are rich in leucine and serine and contain a surprising number of conserved tryptophan residues. However, no obvious motifs or homologies to proteins with known functions were identified. The isolation of cDNA clones for Nup96 that predict a protein product of 186 kD, and the similarity between this protein product and S. cerevisiae Nup145p, prompted us to investigate whether proteolytic processing of the Nup98- Nup96 precursor may also occur. The first approach toward addressing this issue was to determine the NH 2 -terminal residue of Nup96 by direct amino acid sequence analysis. As indicated in Fig. 4 , the NH 2 terminus of Nup96 was found to begin with the serine residue at position 864 of the full-length Nup98-Nup96 precursor. This serine is in exact alignment with a serine at the cleavage site in Nup145p, suggesting that the Nup98-Nup96 precursor is proteolytically processed through a mechanism similar to that occurring with Nup145p in yeast. To further investigate the proteolytic processing of the Nup98-Nup96 precursor and its functional significance, various constructs were designed to express epitope tagged versions of the protein, both in vitro and in vivo . In particular, a mutant of Nup98-Nup96, predicted to be resistant to proteolytic cleavage, was designed using knowledge of mutations made in S. cerevisiae Nup145p . This mutant contains two amino acid substitutions near the predicted cleavage site (F863S and Y866R). Proteolytic processing of the wild-type Nup98-Nup96 precursor was initially assayed using an in vitro transcription/translation system. Consistent with proteolytic cleavage of a precursor protein, two protein products were produced by transcription/translation of the wild-type Nup98-Nup96 precursor in rabbit reticulocyte lysate . The lower band comigrated with the myc-tagged Nup98 domain expressed alone , whereas the upper band migrated with a similar mobility to the myc-tagged Nup96 domain . Although the Nup96 domain has a predicted M r of 96 kD, its mobility by SDS-PAGE is closer to 115 kD. This anomalous mobility is consistent with the mobility of the protein initially identified in the rat liver NPC preparation . These data indicate that rabbit reticulocyte lysate contains the necessary factor(s) for the processing of the Nup98-Nup96 precursor. Therefore, the Nup98-Nup96 precursor with mutations near the cleavage site was transcribed and translated using identical conditions. A protein of ∼210 kD was produced using this mutant, a size consistent with the predicted mobility of the uncleaved precursor . Taken together, these in vitro data support the conclusion that the Nup98-Nup96 precursor is proteolytically processed to generate two nucleoporins, Nup98 and Nup96. To further demonstrate that the cleavage of the Nup98-Nup96 precursor occurs in vivo, the myc-tagged wild-type and mutant plasmids encoding for proteins were transfected into cells in culture. Immunoblot analysis of lysate from cells transfected with the wild-type protein revealed a major band (only the NH 2 terminus of the precursor contains a myc-tag) migrating at ∼100 kD , consistent with the predicted size of the myc-tagged Nup98 domain after cleavage. Analysis of lysate from cells transfected with the mutant Nup98-Nup96 precursor, on the other hand, revealed a major protein of ∼210 kD. These results demonstrate that the Nup98-Nup96 precursor is proteolytically processed in vivo. In S. cerevisiae , synthesis and cleavage of the Nup145p precursor is important for some aspects of targeting the resulting nucleoporins (N-Nup145p and C-Nup145p) to the NPC . To examine the effects and requirements of Nup98-Nup96 precursor synthesis and processing on Nup98 and Nup96 localization, cultured cells were transfected with the various constructs illustrated in Fig. 7 A, and the localization of the expressed proteins was determined by indirect immunofluorescence confocal microscopy. Cells were double labeled with an anti-myc antibody to localize the transiently expressed proteins, and an antibody to Nup358, a marker for NPCs. When the NH 2 -terminal tagged, wild-type Nup98- Nup96 precursor was expressed, labeling of the nuclear envelope was observed, indicating targeting of the cleaved Nup98 domain to the NPC . Labeling of nucleoli was also observed in many cells expressing high levels of protein, possibly a consequence of having saturated binding sites for Nup98 at the NPC or a possible intranuclear localization of Nup98 as previously described . Merging the signals for Nup358 and Nup98 revealed coincident labeling of the nuclear envelope , confirming NPC targeting. The cleavage-deficient mutant of the Nup98-Nup96 precursor was next examined. In this case, only intranuclear labeling was observed, with no evidence of association of the precursor protein with NPCs or nucleoli . Again, cells were double labeled with antibody to Nup358 , and the merged images revealed no detectable overlap between the two signals at the nuclear envelope . This result indicates that cleavage is a prerequisite for NPC localization of Nup98. Expression of the Nup98 domain alone (amino acids 1–863) resulted in a localization that was virtually indistinguishable from the localization observed after expression of the wild-type Nup98-Nup96 precursor, revealing that it could be targeted to the NPC independent of being synthesized as a precursor with Nup96 . However, when Nup96 was expressed independently, a diffuse cytoplasmic signal was detected, with very little if any detectable localization at NPCs . Merging the signal obtained with antibody to Nup358 with the signal for Nup96 revealed some overlap at the nuclear envelope, making it impossible to conclude that the transiently expressed Nup96 was absolutely excluded from NPCs. Relative to Nup98, however, Nup96 is not efficiently targeted to the nucleus, or to NPCs. The cleavage site present in the Nup98-Nup96 precursor protein is also present in the protein predicted by the Nup98-specific transcript . To examine whether the independently expressed Nup98 protein is proteolytically processed, mutations were made near the cleavage site, similar to those made in the Nup98-Nup96 precursor . When a myc-tagged wild-type Nup98 was expressed in rabbit reticulocyte lysate, a protein with a relative molecular mass of 100 kD was observed by SDS-PAGE . This protein product migrated with a similar mobility to the myc-tagged Nup98 product consisting of amino acids 1–863 , suggesting that Nup98 is indeed processed. Expression of the mutant Nup98, resulted in the production of a protein with a relative molecular mass of 110 kD, providing additional evidence that Nup98 is proteolytically cleaved . To determine whether cleavage occurs in vivo, the wild-type and mutant Nup98 proteins were transiently expressed in cultured cells. Immunoblot analysis of cell lysates with an anti-myc antibody revealed proteins of 100 and 110 kD in cells transfected with the wild-type and mutant Nup98 proteins , respectively, indicating that proteolytic cleavage also occurs in vivo. These results indicate that the alternatively spliced Nup98 transcripts each produce identical Nup98 proteins (consisting of amino acid residues 1–863), through proteolytic processing. To determine whether the processing of Nup98 influenced its targeting to the NPC, indirect immunofluorescence confocal microscopy was performed on cells transfected with the wild-type and mutant proteins using the double labeling approach indicated above. The wild-type, cleaved Nup98, was efficiently targeted to the NPC, as demonstrated in Fig. 10 , A–C. Again, labeling of nucleoli was observed in cells expressing high levels of processed Nup98. When mature Nup98 (residues 1–863) was transiently expressed, localization to the NPC was also observed , indicating that mature Nup98 is imported into the nucleus and targeted to the NPC. In contrast, mutant uncleaved Nup98 was found to accumulate strictly in the nucleoplasm, with no detectable association with NPCs . In addition, uncleaved Nup98 was also absent from nucleoli. These data indicate that, as with the Nup98-Nup96 precursor, proteolytic processing of the Nup98 precursor is essential for proper targeting to the NPC. To determine whether Nup96 interacts with other nucleoporins to form specific sub-domains of the NPC, we carried out subfractionation with isolated rat liver nuclei. We identified a sub-complex of the NPC that contains Nup96 and at least five additional proteins . This complex was released from the pore-complex-lamina fraction using heparin and detergent, and then analyzed by sucrose gradient centrifugation. One of the proteins found to cosediment in the gradient with Nup96 had an apparent molecular mass of 105 kD, similar to the expected mass of Nup107 . Immunoblot analysis of the sucrose gradient fractions confirmed that Nup96 and Nup107 cosedimented, peaking in fractions 5 and 6 . In yeast, a similar complex has been observed between C-Nup145p and Nup84p, the putative homologues of Nup96 and Nup107, respectively . This complex also contains a number of additional proteins, including Nup120p, Nup85p, Sec13p, and a Sec13p-related protein, Seh1p. Two of the proteins that we found to cosediment with Nup96 had molecular masses that closely matched the molecular mass of Sec13p (∼35 kD). Therefore, we obtained peptide sequences for these two proteins , to determine their molecular identity. Two internal peptide sequences obtained from the larger of the two proteins indicated that it was identical to the human homologue of yeast Sec13p, a protein referred to as mSec13 or Sec13Rp . Sequence obtained from the second, lower molecular mass protein (referred to as p37) indicated that it was a novel protein with limited homology to Sec13p. Full-length clones coding for this protein will have to be isolated before a complete comparison can be made with Sec13, and with the Sec13-related protein (Seh1p) found to copurify with the homologous yeast complex. In summary, we have identified a complex released from mammalian NPCs that contains Nup96, Nup107, mSec13, and a novel Sec13-related protein. We are using a novel nuclear envelope fractionation procedure to characterize more completely the protein components making up the mammalian NPC. Based on this fractionation procedure and separation of the isolated proteins by reverse phase column chromatography, we estimate that less than one-third of the mammalian nucleoporins have been characterized at the molecular level. This factor contributes significantly to our lack of understanding about how the NPC is assembled, and how it functions to regulate nucleocytoplasmic transport. The fractionation procedure that we have developed yields nucleoporins and NPC-associated proteins in high purity and yield, and has allowed us to identify a significant number of novel proteins. The first NPC-associated protein that we characterized using this fractionation procedure was the SUMO-1 modified form of RanGAP1 . Here, we have reported the characterization of a second protein, a new nucleoporin of 96 kD termed Nup96. The most intriguing aspect of Nup96 is its unusual pathway of biogenesis. Through analysis at a number of levels, we have demonstrated that Nup96 is synthesized as a precursor that is proteolytically cleaved in vivo. Cleavage of this precursor generates not only Nup96, but also the GLFG-containing nucleoporin, Nup98. Nup98 can also be produced independently of Nup96, by what appears to be an alternatively spliced mRNA that does not include the Nup96 open reading frame . Whereas it was not previously recognized that this predicted Nup98 protein is proteolytically processed, we have shown that it is processed like the Nup98-Nup96 precursor. Evidence for the synthesis and processing the Nup98-Nup96 precursor and the Nup98 precursor in vivo are: (a) Northern blot analysis that supports the existence of the precursor mRNAs, and isolation of cDNAs that predict the precursor proteins; (b) NH 2 -terminal peptide sequence of Nup96 that supports the cleavage of the Nup98-Nup96 precursor between phenylalanine 863 and serine 864; (c) mutagenesis of the cleavage sites that yield the uncleaved precursors, both in vitro and in vivo; (d) homology with S. cerevisiae Nup145p, which undergoes posttranslational in vivo cleavage at an identical site . The Nup98-Nup96 precursor is the apparent vertebrate homologue of S. cerevisiae Nup145p. The NH 2 terminus of both proteins, Nup98 and N-Nup145p, are ∼20% identical over their entire length and contain highly conserved GLFG repeats. N-Nup145p is not essential, but it may have RNA-binding activity and function in RNA transport in yeast . In vertebrates, Nup98 is located on the nucleoplasmic side of the NPC, at or near the basket, consistent with a possible role in RNA export . Further supporting such a role, antibodies against Nup98 inhibit export of multiple classes of RNAs when injected into the nuclei of Xenopus oocytes . The COOH-terminal domains of both precursors, Nup96 and C-Nup145p, are also 20% identical over their entire length, but contain no obvious motifs or homologies to proteins of know function. Studies in yeast, nonetheless, indicate a role for C-Nup145p in mRNA export . Like Nup98, Nup96 also localizes to the nucleoplasmic side of the NPC, at or near the basket, consistent with a potential role in mRNA export. Our data obtained from in situ labeling of intact cells indicate that Nup96 may be located near the distal end of the nucleoplasmic basket, in proximity to the site where Tpr associates with the NPC. Of particular interest is the domain around the Nup98-Nup96 cleavage site, as it is especially conserved among protein homologues in yeast, plant, worm, and man. A similar domain is also present at the extreme COOH-terminus of two additional yeast GLFG repeat-containing nucleoporins, Nup100p and Nup116p . In particular, the ∼50 amino acids preceding the cleavage site, and the ∼12 amino acids after the cleavage site, are ∼40% identical between yeast and human (relative to 20% identity between other regions of the proteins), suggesting that this region may be important for the cleavage process. Surprisingly, however, a large portion of this highly conserved domain is not required for proteolytic processing of Nup145p in vivo . This raises the possibility that the domain preceding the cleavage site may have another function, a function that could possibly be regulated by the cleavage process. This domain includes a conserved RNP1 consensus site that has been suggested to contribute to the RNA-binding properties of N-Nup145p . It will be interesting to determine whether the RNA-binding properties of either Nup98 or N-Nup145p are affected by proteolytic cleavage. The conserved residues surrounding the cleavage site itself, H-F-S, do not resemble the consensus site for any known protease, and the exact mechanism for the cleavage process remains to be elucidated. It is also unclear when, or where the cleavage event occurs, although pulse labeling experiments indicate that the Nup98-Nup96 precursor is cleaved within five minutes of being synthesized (data not shown). Whereas the physiological relevance of producing both Nup98 and Nup96 through proteolytic cleavage of a precursor is not known, the conservation of this pathway from yeast to human suggests a potentially important function. Posttranslational proteolytic cleavage of precursor proteins is widely used to regulate many different cellular functions. Processing of many neuropeptides, hormones and certain plasma proteins is used to regulate the formation of mature, active factors without the need for de novo protein synthesis . Protelolytic cleavage can also function to indirectly activate proteins by regulating their localization within the cell. Examples include the signal-mediated nuclear targeting of Notch-1 from the plasma membrane , and relocalization of the sterol regulatory element binding proteins from the nuclear membrane to the nucleus in response to sterol levels . Other possible functions of precursor synthesis and proteolytic cleavage include protein folding, as has been suggested for certain ribosomal protein-ubiquitin precursors , and as a mechanism to strictly control protein stoichiometry. Another important role for regulated proteolytic cleavage is in the assembly and maturation of large molecular complexes, best exemplified by virus particle assembly . By this analogy, proteolytic cleavage of nucleoporins could be related to the orderly assembly of the NPC. As an example, the Nup98-Nup96 precursor could be inserted into a newly synthesized pore and cleaved only after the appropriate cleavage factor is assembled. Cleavage may then result in conformational changes, or exposure of binding sites on Nup98 and/or Nup96, that allow other sets of proteins to bind in an orderly sequence. Our data demonstrate that proper processing of both the Nup98-Nup96 precursor and the Nup98 precursor is required for efficient NPC association, indicating that there is an ordered series of events leading to the association of these nucleoporins with the NPC. Mutations in the cleavage site that prevent processing lead to accumulation of both proteins in the nucleoplasm, but not at NPCs. In the case of Nup98, this result suggests that the COOH-terminal 58 amino acids (that would normally be removed) either tether Nup98 to intranuclear sites, or mask the NPC-targeting domain. Interestingly, in addition to not being targeted to NPCs, unprocessed Nup98 was also absent from nucleoli when over expressed. Although the significance of processed Nup98 appearing in nucleoli is not clear, it has previously been suggested that Nup98 may not be absolutely confined to NPCs . These data raise the intriguing possibility that Nup98 could shuttle between nucleoli and NPCs. In addition to cleavage being important, our studies also demonstrate that proper targeting of Nup98 and Nup96 to the NPC is influenced by the synthesis of the precursor protein. In particular, we found that targeting of Nup96 to the NPC is dependent on its being synthesized as a precursor. When expressed independently, Nup96 accumulates in the cytoplasm, suggesting that its nuclear import may be Nup98-mediated (Nup98 expressed alone is imported into the nucleus). Nup98-mediated import of Nup96 could occur in one of two ways. Either the Nup98-Nup96 precursor could be imported into the nucleus before proteolytic processing, or Nup98 and Nup96 could remain associated after proteolytic processing in the cytoplasm, and then be imported as a complex. At present, there is no data to suggest that Nup98 and Nup96 remain associated with each other once they are inserted into the NPC. The sub-complex of nucleoporins containing Nup96, Nup107, and mSec13 that we isolated does not contain Nup98, and experiments in yeast indicate that the analogous sub-complex also lacks N-Nup145p . To date, relatively few sub-complexes of the mammalian NPC have been identified. And although strikingly similar at a morphological level , no homologous sub-complexes of mammalian and yeast NPCs have previously been identified. This has raised the notion that the molecular structure of the NPC may be less well conserved than its morphological structure. We have identified a complex of mammalian nucleoporins that contains Nup96, Nup107, mSec13, at least one Sec13-related protein, and at least two additional proteins of ∼150 kD. This complex is highly similar to the Nup84p complex of yeast, and therefore represents one of the first homologous complexes of nucleoporins identified between yeast and mammals. There is also evidence for homology between a yeast nucleoporin complex containing Nup159p, Nup82p, and Nsp1p and a vertebrate complex containing Nup214/CAN, Nup88, and possibly p62 . The identification of these complexes suggests that the molecular structure of the NPC may be more highly conserved than was anticipated. Similarly, homologous sub-complexes will likely be identified in the future, as more mammalian and yeast nucleoporins are identified and characterized. In the immediate future, further purification of this complex will allow us to identify several additional nucleoporins, and possibly obtain information about its structural features. Of particular interest is the presence of the Sec13-related proteins in this complex. Whereas the exact role that Sec13 may have at the NPC has been previously discussed , our combined data suggest that this function is likely to be exerted on the nucleoplasmic side of the NPC, given the localization Nup96. It will be interesting to investigate the role that Sec13 may have in NPC biogenesis using Xenopus nuclear envelope reconstitution systems. What are the functions of Nup98 and Nup96 once assembled into the NPC? As indicated, both Nup98 and Nup96 localize to the nucleoplasmic side of the NPC, at or near the basket. The nucleoplasmic basket appears to have an active role in binding RNP complexes and mediating their export from the nucleus. Electron micrographs of Balbiani ring RNPs being transported through the NPC show intimate interactions between the basket and the RNP, implying that the proteins in this structure actively engage the particles during transport . It is not yet known whether the nucleoporins at the basket bind the RNA itself, and/or the hnRNP proteins that accompany the RNA during transport . As implied from their localization, and the functional studies of Nup145p in yeast and Nup98 in Xenopus , Nup98 and Nup96 are likely to be important factors mediating the docking and translocation of RNPs through the NPC. The challenge ahead will be to understand how these nucleoporins interact with RNPs as they are transported through the NPC, and how these interactions regulate the transport process. | Study | biomedical | en | 0.999998 |
10087257 | H-2K b clone 18, a cloned, conditionally immortal myogenic line, was derived from a 4-wk-old male H-2K b -tsA58 heterozygote, carrying a single copy of the thermolabile tsA58 mutant SV-40 large T antigen (TAg) under the control of the H-2K b promoter . The line was maintained at low density in DME (high glucose, with sodium pyruvate; Life Technologies Inc.) supplemented with 20% FCS, 2% chick embryo extract, 4 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin ( Sigma Chemical Co. ). Murine recombinant IFN-γ was added to the medium (20 U/ml; Life Technologies Inc.) to increase transgene expression through the H-2K b promoter, and cultures were grown at 33°C in 10% CO 2 , a temperature at which the thermolabile TAg protein is active. Primary MPCs were prepared by enzymatic disaggregation of leg muscles from 1-d-old male C57Bl/10 mice. Muscles were minced and digested in HBSS (25 ml/mouse) containing 1 U/ml collagenase type III ( Sigma Chemical Co. ) for 10 min at 37°C before repeated cycles of aspiration and expulsion through the tip of a Pasteur pipette. Released cells were discarded and fragments were subjected to two rounds of digestion in HBSS containing 0.25% trypsin ( Sigma Chemical Co. ) as above. The trypsin was inactivated by addition of an equal volume of HBSS containing 20% FCS, and cells were collected by centrifugation at 350 g for 10 min at 4°C. Cell pellets were resuspended in growth medium consisting of DME (high glucose, with sodium pyruvate) supplemented with 20% FCS, 2% chick embryo extract, 4 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Undigested tissue fragments were removed by filtration through 45 μM-pore-diameter nylon mesh, and cells were plated and cultured at 37°C in 10% CO 2 . MPCs were radiolabeled by culturing for 16 h in growth medium containing 0.25 μCi/ml [methyl- 3 H]thymidine (5 Ci/mmol) or [methyl- 14 C] thymidine (54 mCi/mmol; Nycomed Amersham ). Using this labeling regime, >95% of the radiolabel could be TCA-precipitated (data not shown). To investigate the effects of radiolabeling on MPCs, [ 14 C]thymidine–labeled, [ 3 H]thymidine–labeled, and unlabeled H-2K b clone 18, MPCs were plated at 500 cells/cm 2 . At each time point, cultures were examined by phase-contrast microscopy and total cell number was calculated from mean number of cells in 12 random fields of known area. H-2K b clone 18 MPCs were plated at 500 cells/cm 2 , cultured for 16 h in growth medium containing 4 μM BrdU, and then immunostained with mouse anti-BrdU antibody as described . In brief, cultures were fixed at −20°C in 95% ethanol, rinsed in PBS, and then incubated for 30 min at room temperature in 2 M HCl. After three 20-min washes in 50 mM NaCl, 100 mM Tris-HCl, pH 7.4, the cells were incubated for 1 h with mouse anti-BrdU antibody (diluted 1:20 in PBS), for 30 min with a biotinylated rabbit polyclonal anti–mouse immunoglobulins antibody (diluted 1:250 in PBS), and finally for 30 min with streptavidin-peroxidase (diluted 1:250). All antibodies were obtained from DAKO Corp. Peroxidase activity was visualized with 3,3′-diaminobenzidine tetrahydrochloride and after counterstaining with hematoxylin, cultures were examined by light microscopy. Percentages of BrdU-positive cells were counted in six random fields, each containing ≥200 cells. MPCs were detached from the culture vessel by incubation in 0.05% trypsin/0.02% EDTA. An equal volume of growth medium was added to inactivate the trypsin, and cells were collected by centrifugation at 350 g for 10 min at 4°C. Cell pellets were resuspended in HBSS such that 5 × 10 5 cells could be transplanted in a total volume of 5–10 μl. 3–4-wk-old female mdx nude mice were anaesthetized, the skin overlaying the tibialis anterior (TA) muscle was opened, and 5 × 10 5 MPCs were injected into the TA muscle using a Hamilton 7005 syringe. The needle was inserted longitudinally into the muscle and withdrawn slowly as the plunger was depressed to deliver the cells along the length of the muscle. Irradiated host muscles had been exposed to 18 Gray of X-irradiation 3 d before transplantation, a pretreatment previously shown to enhance MT in mice . At the indicated time points, host muscles were removed, snap-frozen in liquid nitrogen, and stored at −80°C. Amounts of radiolabel and Y chromosome were measured as described previously . In brief, muscles were thawed on ice, minced, and then digested to homogeneity for 16 h at 50°C in 50 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, pH 8.0, containing 500 μg/ml proteinase K ( Sigma Chemical Co. ), and 0.5% (wt/vol) SDS, with regular vortexing. To determine the amount of radiolabel present, an aliquot was mixed with Hionic-Fluor liquid scintillation counting fluid (Canberra Packard Ltd.) and the amount of isotope measured using a Beckman LS6000SC counter system. DNA was extracted from the remaining digestion mixture and slot blotted onto uncharged nylon membrane (Hybond-N; Nycomed Amersham ), together with appropriate control dilution series of male and female DNA. Before slot blotting, the amount of isotope present in an aliquot of each DNA preparation was measured as above to determine the efficiency of extraction. Slot blots were hybridized with a Y chromosome–specific DNA probe, 145SC5 , and labeled with [α- 32 P]dCTP (Nycomed Amersham ) by random priming using Ready-To-Go DNA labeling beads ( Pharmacia Biotech Co). Hybridized membranes were exposed to a phosphor screen which was then scanned using a PhosphoImager 445 SI (Molecular Dynamics, Inc.). Quantitative analyses were carried out using ImageQuant software (Molecular Dynamics, Inc.) and the amount of male DNA present in each sample was determined by reference to a standard curve obtained from control male DNA dilution series. In the experiment presented in Fig. 2 , host muscles were removed, solubilized in Soluene ® -350 (Canberra Packard Ltd.), and the amount of 14 C present was measured by scintillation counting after the addition of scintillation counting fluid, as above. 8-μm-thick cryosections were cut and immunostained for dystrophin using a sheep polyclonal antibody to the 60-kD dystrophin peptide (provided by Professor E.P. Hoffman, University of Pittsburgh School of Medicine, Pittsburgh, PA) as described . In brief, unfixed sections were blocked in 5% horse serum in PBS, and then incubated for 1 h with the primary antibody (1:1,000 dilution), for 1 h with an affinity-purified, biotinylated donkey antibody raised against sheep immunoglobulins (diluted 1:400; Nycomed Amersham ), and for 30 min with streptavidin– Texas red (diluted 1:100; Nycomed Amersham ). All dilutions were in 5% horse serum in PBS. The slides were examined and photographed using a Zeiss Axiophot fluorescence microscope. Initial studies were carried out using a cloned, conditionally immortal myogenic cell line, H-2K b clone 18, derived from a male mouse transgenic for the tsA58 mutant TAg under the control of the inducible H-2K b promoter . Donor MPCs were labeled with either [ 3 H]thymidine or [ 14 C]thymidine, and then injected into preirradiated TA muscles of 3–4-wk-old female nude mdx mice, an optimized host environment for MT . Identical results were obtained with [ 3 H] and [ 14 C]thymidine–labeled MPCs . In the experiments shown, each of which was carried out using a different preparation of H-2K b clone 18 MPCs, there was a rapid loss of 80–95% of radiolabel and Y chromosome during the first day after transplantation (at 24 h, mean percentage of radiolabel remaining from all experiments = 10.2 ± 4.2; mean percentage of male DNA remaining = 11.2 ± 1.6 [± SEM, n = 12]). During this period, the loss of the two markers was concordant. The mean ratio of percentage of radiolabel to percentage of male DNA present at 24 h remained at 0.96 ± 0.04 (± SEM, n = 12) compared with the initial ratio of 1.0. Equivalent loss of Y chromosome demonstrates that the disappearance of radiolabels was not due to leakage of unincorporated thymidine. Furthermore, in some experiments, entire legs of host mice were solubilized after removal of the injected TA muscles, and were found to contain <10% of the injected isotope 4 h after MT (data not shown). This, together with the finding that MPCs incorporated within a fibrin clot are lost to the same extent as when delivered in suspension , demonstrates that the disappearance of donor cells is not simply due to leakage from the injection site into surrounding compartments. The rapid disappearance of donor MPCs from host muscles was confirmed and further defined by a detailed investigation of the first 24 h after MT . Muscles harvested immediately after injection contained 85% of the amount of label present in the transplanted cell population, confirming that most of the cells were successfully deposited within the muscle. However, only 30% of the injected label (∼35% present immediately after injection) remained after 1 h. Following the initial fall, amounts of radiolabel present in muscles taken between 1 and 8 h after injection were relatively constant, at ∼25% of that transplanted. However, a second loss of label was observed beginning 8–12 h after transplantation, suggesting a biphasic loss of donor MPCs following MT: α rapid loss of ∼75% of the injected cell population, followed by a second event beginning 8–12 h later, result in the loss of most of the remaining cells, such that only ∼1% of the injected radiolabel remained 4 d after grafting . After the dramatic disappearance of donor cells during the initial 24 h after injection, there was a progressive loss of radiolabel such that ∼1% of the injected radiolabeled-thymidine remained 4 d after transplantation . This value may actually overestimate the persistence of the transplanted MPCs as some label may have been released by dying cells and reused by host cells. However, continued disappearance of donor cells was accompanied by division of the remaining cells, as revealed by a marked divergence of the labels. Thus, although only 1% of the injected radiolabel persisted, the mean percentage of Y chromosome present after 4 d from all experiments was equivalent to 23.5% of the initial population (SEM ± 3.8, n = 12). It is notable that, although the percentages of Y chromosome present in individual muscles after 4 d varied from 8 to 43%, the ranges within each experiment were small , demonstrating that within an equivalent group of host mice, each particular cell preparation gave rise to equal numbers of donor cells irrespective of the radiolabel used. Furthermore, when Y chromosome was measured in muscles transplanted with unlabeled MPCs, the same results were obtained as with equivalent labeled MPCs . Extensive proliferation of the surviving fraction of donor cells and their subsequent contribution to the formation or repair of muscle fibers was also evident from the large numbers of dystrophin-positive profiles present in muscles taken 3 wk after MT . Again, no differences in the numbers or distributions of dystrophin-positive fibers were observed between muscles that had received [ 3 H]thymidine–labeled, [ 14 C]thymidine–labeled, or unlabeled MPCs. Together, these results demonstrate what would appear to be a catastrophic loss of donor cells in the immediate aftermath of MT, cited as an explanation for failure of this technique , is in fact a standard feature of successful transplantations, as in the above experiments, and that success is due to extensive proliferation of a small proportion of the cells rather than survival of the majority of the transplanted population. It was surprising that identical results were obtained in vivo with MPCs labeled with [ 3 H]thymidine and [ 14 C]thymidine . In vitro, [ 3 H]thymidine was cytotoxic for the majority of the population, whereas [ 14 C]thymidine had no significant effect on survival or proliferation . Previous studies, published >30 yr ago, reported that [ 3 H]thymidine of high specific activity, such as used in the present studies, was cytotoxic to HeLa cells, and only cells which had not entered S phase during the period of labeling (and therefore remained unlabeled) retained the potential to form colonies . Since intranuclear [ 3 H]thymidine is cytotoxic to MPCs in vitro, this suggests that cells that survive MT and proliferate within the host muscle environment had not undergone DNA synthesis during the 16 h of radiolabeling, and may therefore represent a subpopulation of MPCs defined by a relatively slow generation time in vitro. When H-2K b clone 18 MPCs were cultured under the same conditions used for radiolabeling, but in the presence of BrdU, immunostaining for BrdU incorporation revealed that 3 ± 0.3% (± SEM, n = 8) of the population had not entered S phase during that time, suggesting an upper limit to size of the subpopulation responsible for muscle formation in vivo. The preceding data are from experiments in which MPCs were injected into preirradiated host muscles, a treatment previously shown to increase the efficacy of MT . To investigate the effects of preirradiation of host muscles on the short-term behavior of transplanted MPCs, the right legs of a series of female nude mdx mice were irradiated before bilateral MT with radiolabeled primary cells derived from male neonatal muscle. In the irradiated muscles and the contralateral, nonirradiated controls, there was a similar loss, both in timing and extent, of virtually all the transplanted radiolabel over the experimental period . As with the conditionally immortal cell line, the amount of Y chromosome showed a progressive relative increase from 24 h after MT onwards compared with the amount of radiolabel, in both the irradiated and control muscles. However, the amount of Y chromosome increased to a greater extent in the irradiated muscles, suggesting that there was greater proliferation of surviving MPCs than in nonirradiated muscles. Thus, the increased efficacy of MT in preirradiated host muscles is not the result of increased cell survival, but may be attributed to increased proliferation of the surviving subpopulation. Furthermore, data presented in Fig. 5 also show that successful MT through the rapid expansion of a small number of surviving MPCs is not peculiar to conditionally immortal myogenic lines. In fact, when injected into irradiated host muscles, more donor-derived nuclei were generated from primary cells than from the same numbers of cloned, conditionally immortal MPCs, although a similarly large proportion of cells was lost in the immediate aftermath of transplantation. Successful genetic modification of skeletal muscle by MT requires the survival of donor myoblast nuclei and their stable incorporation into muscle fibers within the host tissue. Attempts to develop MT into a viable approach to gene therapy have focused on modification of the host tissue and control of inflammatory and immunological responses to the graft . In contrast, scant attention has been paid to donor cells until recently . In the context of MT, the term myoblast is simply used to describe a mononucleated, undifferentiated cell with the potential to initiate the myogenic program, withdraw from the cell cycle, and differentiate to form skeletal muscle. However, our studies of early events following MT reveal markedly divergent behavior within myoblast populations, even in X-irradiated muscles of nude mdx mice, a highly permissive environment for MT . The vast majority of donor MPCs die in the aftermath of MT, and muscle of donor-origin is derived from only a tiny proportion of the injected population which undergo rapid and extensive proliferation in vivo. This phenomenon was observed with both primary MPCs and a myogenic cell line derived from the H-2K b -tsA58 transgenic mouse, suggesting that expression of TAg maintains the population capable of repopulating muscle in vivo. To follow the fate of donor myoblasts during the first few days after transplantation into dystrophic host muscle, we have developed a method based upon quantitative comparisons between a genetic marker (Y chromosome) and a semiconserved label (radiolabeled thymidine) of donor DNA . Since both labels are measured directly, this combination has an advantage over other commonly used reporter systems such as β-galactosidase and green fluorescent protein, the use of which may be confounded by variation in the level of gene expression or accumulation of product, particularly when labeled cells may be at different stages of differentiation. Loss of either label will occur only on cell death (loss of radiolabeled thymidine due to DNA repair would be trivial in the time course of this experiment). Thus, in our system, the amount of Y chromosome provides an absolute measure of the number of copies of donor cell–derived genome, while the amount of radiolabel directly reflects the proportion of originally grafted donor cell DNA which has survived in the recipient muscle. Our intention to use the change in ratio between the genetic and semiconserved labels to assess cell proliferation is predicated on uptake of radiolabeled thymidine by a single homogeneous population. However, the almost complete loss of radiolabel in conjunction with increasing amounts of Y chromosome revealed that labeling was not homogenous and suggested the presence of two donor cell populations of diverse behavior. One, proliferative in culture and therefore readily poisoned by [ 3 H]thymidine, is sensitive to transplantation into muscles. The second, a minor subpopulation, slowly dividing in culture and thus refractory to [ 3 H]thymidine, survives transplantation and proliferates rapidly in vivo. This unexpectedly clear-cut distinction, to some extent, vitiates the use of the ratio of labels as a quantitative measure of cell proliferation. Thus, with progressive decline of radiolabel towards background levels, the best indicator of the behavior of the surviving subpopulation is the change in the absolute measures of Y chromosome content which, for example, illustrates a clear difference between the rapid proliferation of cells in preirradiated muscles and slower rates in nonirradiated muscles. Our findings raise two fundamental questions. Why should a consistently small, but highly prolific, subpopulation of donor MPCs survive the apparent rigors of MT, and what causes the death of the vast majority of transplanted MPCs? The identity of cells which proliferate and contribute to muscle formation is crucial to the first of these questions. Our data suggest that the surviving cells constitute a distinct minority which, for unknown reason(s), are resistant to the early events after MT, and are not merely chance survivors of a purely stochastic process of cell death. We observed the same dynamics of rapid cell death and eventual outcome of MT in terms of new muscle formation with unlabeled, [ 14 C]thymidine–labeled, or [ 3 H]thymidine–labeled MPCs, despite the fact that the latter label is cytotoxic to dividing cells. Cells which proliferate after transplantation and give rise to the donor muscle therefore must have been those that are refractory to [ 3 H]labeling, presumably because they had not passed through S phase during the 16-h period of in vitro labeling. That the loss of injected MPCs was identical irrespective of labeling suggests that cells in rapid cycle in vitro (which are readily labeled) are those which die following MT, and are irrelevant to the final outcome. Further, this demise is an innate feature of this category of cell since its occurrence is unaffected by the toxicity of the label. In contrast, the cells which do not incorporate radiolabel in vitro further demonstrate their inherent behavioral individuality by undertaking rapid proliferation soon after transplantation into irradiated muscle, in conditions which the previously proliferative majority are dying. These findings parallel those in heterogeneous populations of hematopoietic cells, where exposure to high specific activity [ 3 H]thymidine has been shown to selectively kill cycling cells while preserving long-term culture-initiating cells , and argue in favor of the idea that cells which give rise to muscle on transplantation into muscle constitute a distinct subpopulation, apparently with some stem cell–like properties. The existence of a stem cell–like subpopulation of MPC has also been suggested by recent studies of myoblasts in tissue culture. Yoshida et al. identified a population of reserve cells in the myogenic cell line, C2, which, when exposed to differentiation conditions, persisted as slowly cycling, undifferentiated, mononuclear cells with the capacity to self-renew and give rise to differentiating cells. Phenotypically distinct, nonfusing cells that can divide asymmetrically to self-renew and generate committed cells have also been identified within individual clones of human MPCs . These presumptive stem cells comprised ∼2% of each clone (very similar to our 3% upper limit of murine muscle-forming MPCs which did not enter S phase during labeling) and decreased in number with successive passages, which may account for the finding that culture of avian myoblasts results in a progressive decrease in the amount of muscle formed following transplantation . Recently, Dominov et al. reported that Bcl-2, an apoptosis-inhibiting protein, is expressed by myoblasts at an early stage of differentiation and may promote clonal expansion of myogenic cells. Intriguingly, only 1–4% of cells in primary myogenic cultures were found to be positive for Bcl-2 . It is possible that the subpopulation with some stem cell–like properties, previously identified in vitro , may correspond to those we define by their behavior in vivo, i.e., relative robustness when transplanted. Why most donor MPCs die following MT remains unclear. Our results show that ∼99% of radiolabeled donor cells had died and were cleared from the host muscles 4 d after grafting. The remaining 1% defines an upper limit to the survival of radiolabeled cells, as some reutilization of label released from dead donor MPCs may have occurred, although the extent of any reutilization was clearly insignificant compared with the total amount of label transplanted. We found no evidence of donor cell apoptosis during the first 6 h after MT when sections of host muscle were analyzed by terminal deoxynucleotidyl transferase– mediated deoxyuridine triphosphate endlabeling, a technique which reveals DNA strand breaks characteristic of apoptotic nuclei (data not shown). The mechanism is also unlikely to be a specific immunological response to donor MPCs as death occurs within 2–3 d following either transplantation into isogenic , congenic , immunodeficient, or immunosuppressed hosts . A recent study has implicated a nonspecific inflammatory reaction, possibly initiated in response to tissue damage, which may be inhibited by antibody-mediated blockade of leukocyte function associated molecule-1 . Our analysis of the first 24 h after MT suggests that the rapid loss of cells may involve two events, the second of which, beginning 8–12 h after injection, may indeed be attributable to such an inflammatory reaction. However, this is unlikely to account for the initial loss of ≤70% of donor cells within 1 h of transplantation. Rather, this rapid necrotic demise of the most proliferative portion of the population of grafted MPCs suggests the cells are maladapted to some aspect of the environment within the recipient muscle, perhaps anoxia, although the rapid removal of released radiolabel from the graft site seems to imply that it is efficiently perfused. In the study of Guérette et al. , the percentage of donor cells surviving 3 d after grafting was calculated relative to that measured 1 h after MT. Our results show that significant cell death occurs during the first hour after grafting, so the initial phase of cell death following grafting would have been missed. However, recent findings of Qu et al. also imply a biphasic loss of donor MPCs following MT. When either an mdx cell line or myoblasts isolated from isolated muscle fibers were grafted, substantial losses of donor cells were measured 12 h after injection. In marked contrast, primary myogenic cells selected by a sequential preplating regime survived transplantation, apparently without loss. However, although the selected primary cells persisted for 48 h, there were marked losses during the subsequent 3 d. Furthermore, when the mdx cell line was engineered to produce IL-1 receptor antagonist protein intended to block any early inflammatory reaction, the initial loss of donor cells was largely unaffected, although the subsequent disappearance observed 2–5 d after transplantation was prevented . These results support the idea that loss of donor MPCs following MT involves two events, the first as yet undefined and to which different preparations of donor MPC may be differentially susceptible, followed by a second, inflammation-mediated event. The identification of a relatively undifferentiated subpopulation, slowly dividing (and probably selected against) in culture, but driven into rapid proliferation upon being grafted into preirradiated muscle, and in consequence responsible and therefore required for successful MT in vivo, has significant implications for human MT. Failure to reproduce the promising results obtained with mdx mice in clinical trials of MT on boys with Duchenne muscular dystrophy has been variously attributed to lack of host muscle regeneration, the presence of significant connective tissue barriers (both consequences of recipient age), and immune rejection . The ability of human MPCs to survive transplantation was also questioned and subsequent reports of rapid donor cell death during the aftermath of MT in mice have suggested that this may have contributed to the failure of clinical trials. Our results also demonstrate that the vast majority of transplanted MPCs die within hours of delivery. However, we have shown that this phenomenon does not lead to inevitable failure, as it occurs during successful MT. Recent reevaluation of biopsies of recipient muscles from an earlier clinical trial of human MT on boys with Duchenne muscular dystrophy revealed the persistence of unexpectedly large numbers of donor-derived nuclei, many of which had become incorporated into mature myofibers, but did not express dystrophin . Therefore, at least in human MT, the ability of donor cells to survive grafting does not inevitably lead to biochemical modification of host tissue, possibly due to environmental influences encountered in diseased, dystrophic muscle. Nevertheless, the ability to isolate and maintain a population of human MPCs equivalent to those responsible for successful MT in mice could greatly enhance the therapeutic potential of a given dose of donor cells, and might obviate the extensive cloning and expansion in vitro , or selection on the basis of neural cell adhesion molecule expression , which may have helped to confound the potential success of MT in human trials. Considerable interest in the possible use of stem cells for genetic conversion of adult skeletal muscle has resulted from the demonstration that bone marrow–derived cells can contribute to skeletal muscle regeneration, following either direct grafting into host muscle or bone marrow transplantation . Whether derived from bone marrow or isolated from heterogeneous myogenic cultures , precursor cells with the potential to contribute to new muscle formation, particularly after the initial transplantation, would be invaluable for the development of MT. | Study | biomedical | en | 0.999999 |
10087258 | The genetic markers and chromosomes used for mutagenesis and mapping are described in Lindsley and Zimm . Most stocks were from the Bloomington Drosophila Stock Center, while the l(2)k06308 and l(2)k05318 strains were provided by the Berkeley Drosophila Genome Project Stock Center. The mfl 1 allele was isolated in a small-scale P-element mutagenesis screen performed essentially according to the “reversion jumping” scheme . In our experiments, toc l(2)01361 , a lethal P[LacZ, ry + ] insertion at the toucan locus, was mobilized by the P[ry + , Δ(2-3)99B] element as a source of transposase. Males carrying both the Δ(2-3) and toc l(2)01361 elements were crossed to females carrying a lethal toc allele that lost the ry + marker. This allele, named toc Δ01361 , was generated in our laboratory from toc l(2)01361 by P imprecise excision. In the next generation, flies lacking the CyO chromosome balancer (reversion event of the toc l(2)01361 allele) but marked with ry + were recovered, and second chromosomes carrying these new insertions were balanced and retained for further study. Single P-element insertions were verified by genomic Southern blot analyses with PZ-derived probes. Wild-type P-element excised revertants were generated by crossing homozygous mfl 1 males to w 1118 ; CyO /L 2 ; Sb, P[ry + ,(Δ2-3)99B]/TM6B,Tb virgin females and by individual mating of disgenic F 1 males to 5-10 CyO /Sp; ry 506 virgin females. Individual non-Stubble males that lost the ry + marker were collected from the F 2 progeny and balanced over the CyO chromosome. The resulting stocks were checked for the presence of homozygous revertant flies in which P-element excision was verified by PCR amplification and DNA sequence analysis. In situ hybridization to salivary gland polytene chromosomes was performed with a DIG-labeled probe derived from the PZ element, essentially as described in Ashburner . The P-element hsp70:mfl construct (P[ hs:mfl ]) used for P-element– mediated transformation was prepared by inserting a 1833 bp cDNA sequence containing the complete mfl ORF into the EcoRI site of the pCaSpeR-hs-act vector . Transgenic flies carrying the P[ hs:mfl ] on the X or third chromosome were used to introduce the transposon in mfl mutant background. Lethal phase analysis was performed according to Fletcher et al. . As control, lethal phases of mfl / Df(2R)Px4 transheterozygous were also determined. To identify homozygotes carrying mfl lethal alleles we generated y w ; mfl / CyO y + and y w P[ hs:mfl ]; mfl/CyO y + stocks in which homozygous mutant larvae were distinguished from their mfl / CyO y + heterozygous siblings by the yellow phenotype of mouth hooks and denticle belts. Basic cloning techniques, DNA and RNA extraction, manipulation and labeling, screening and sequencing techniques were carried out according to Sambrook et al. . For Northern blot analysis, 5 μg of poly(A) + or 10 μg of total RNA were electrophoresed and transferred to Hybond-NX ( Amersham ) filters for hybridization. The 5′ end of SnoH1 RNA was determined by primer extension analyses, using 50 μg of total RNA together with primers complementary to nucleotides 96–135 and 149–189 of the fourth mfl intron. rRNA processing was studied by [ 3 H]uridine (1 mCi/ml, 22.4 Ci/nmol) incorporation in Drosophila larvae. After 48 h, total RNA was extracted and analyzed by agarose electrophoresis followed by fluorography, as described by Tollervey . In rRNA northern blot analyses, probe I corresponds to oligonucleotide 5′-GTTAAAATCTTTTTATGAGGTTGCCAAGCCCCACAC-3′; probe II to oligonucleotide 5′-CACCATTTTACTGGCATATATCAATTCCTTCAATAAATG-3′; probe III to oligonucleotide 5′-CTATTTCCGAATCATTAATAAGAGACAATTCTAGATG-3′. Mapping of Drosophila ribosomal pseudouridines was performed essentially as described by Bakin and Ofengand using as primer the oligonucleotides: 5′-AATCAAGTTCGGTCAACTTTTGCGAAACAACCGTAACAC-3′ for 18S U1820, U1821, and U1822; 5′-GCGTCGTAATACTAATGCCCCCAAACTGCTTC-3′ for 18S U830/U831, U840, U841, and U885; 5′-CCATTCATGCGCGTCACTAATTAGATGACGAG-3′ for 28S U2442, U2444, and U2499. Western blots were analyzed with a 1:1,000 dilution of an affinity-purified rabbit anti-MFL antibody, kindly provided by S. Poole (University of California, Santa Barbara, CA). Whole mount ovaries in situ hybridization, using single-stranded DIG-labeled probes, obtained by PCR, and immunohistochemical staining of ovaries were performed essentially as described in Ashburner . The rabbit primary anti-MFL antibody, kindly provided by S. Poole, was diluted 1:400 and detected with a biotin-conjugated secondary antibody and a horseradish peroxidase–biotin–avidin complex (ABC Elite Kit; Vector Labs). Sequence comparisons were performed using the BLAST search algorithms available at the National Center for Biotechnology Information Web pages; multiple alignments were performed using the CLUSTAL and BOXSHADE programs. The snoH1 RNA putative secondary structure was established using the MFOLD program. The first minifly allele, mfl 1 , was isolated in our laboratory in the course of a PZ-element mutagenesis screen on the second chromosome (see Materials and Methods) as a viable, recessive mutation causing a variety of phenotypic abnormalities. The mfl 1 pleiotropic phenotype included an extreme reduction of body size , developmental delay, essentially due to a 4–5-d prolongation of the larval life, defects in the abdominal cuticle , strong reduction in the length and thickness of abdominal bristles, and reduced female fertility. Most traits of the mfl 1 phenotype largely overlapped those caused by the Drosophila Minute , mini or bobbed mutations that affect, respectively, the synthesis of ribosomal proteins, 5S, or 18S and 28S rRNAs. This similarity suggested for mfl a possible role in ribosome biogenesis, encouraging us to attempt the molecular cloning of the gene. mfl 1 mutation was caused by a single P-element insertion, which, by in situ hybridization of a P-specific probe to salivary gland polytene chromosomes of mfl 1 heterozygous larvae , was mapped on the chromosome arm 2R, at the 60B-60C polytene subdivisions boundary. Given that wild-type revertants could be recovered from dysgenic crosses after precise excision of the element (see Materials and Methods), mfl 1 mutation appeared to be directly caused by this single PZ insertion. Complementation analysis assigned the gene to the region covered by the Df(2R)Px4 deficiency. Among a number of P-induced lethal mutations recovered by Török et al. and subsequently deposited as part of the Berkeley Drosophila Genome Project, five mapped at the 60B-60C polytene subdivisions boundary. These mutations were all tested in a complementation analysis, by crossing each of them to mfl 1 heterozygous flies. Two lines, l(2)k05318 and l(2)k06308 , yielded transheterozygous flies with a strong mfl phenotype at the expected ratio, leading us to conclude that they belonged to the mfl complementation group and represented lethal mfl alleles. Accordingly, these lines were renamed, respectively, mfl 05 and mfl 06 . Previous cytological mapping by the Berkeley Drosophila Genome Project assigned these two mfl alleles to the polytene interval 60B11-C2, in good agreement with our results. By lethal phase analysis we observed that mfl 05 homozygotes die mainly as first instar larvae, while most of the mfl 06 animals die later, either as second or mainly as young third-instar larvae. Both mfl 05 and mfl 06 animals fail to increase their size as compared with their wild-type heterozygous siblings and survive for an additional 4–5 d as first or third instar larvae, respectively. Since a feature of the mfl 1 pleiotropic phenotype was represented by reduced female fertility, we looked at the structure of mutant ovaries. Morphological abnormalities were often observed, with some of the egg chambers beginning to degenerate beyond approximately stage 7 of oogenesis . In the degenerating egg chambers, fragmented or condensed nurse cell nuclei with irregular shape are frequently found . These observations raise the possibility that apoptotic cell death may occur in mfl 1 abnormal ovaries. This possibility was investigated by staining egg chambers with acridine orange (AO). AO is a vital dye that is known to selectively stain apoptotic cells in insects and has successfully been used to study the distribution of apoptosis in Drosophila ovaries . In our experiments, wild-type ovaries exhibit a diffuse green fluorescence , whereas highly fluorescent yellow spots are detected in mfl 1 degenerating egg chambers . These yellow spots are known to correspond to apoptotic, AO highly positive nuclei , thus confirming the occurrence of apoptosis in mfl 1 ovaries. As a consequence of the gonadic abnormalities observed, mfl 1 homozygous females lay a reduced number of mature eggs, and ∼15% of the embryos produced failed to hatch. Such degenerating embryos show asynchronous and atypical development, invariantly accompanied by diffuse apoptotic cell death (data not shown). Many mutations causing partial loss-of-function of vital genes interfere with the proper development of the egg, causing female sterility. Inadequate rate of protein synthesis is also known to affect Drosophila oogenesis, by slowing the level of yolk production and retarding egg chamber progression into vitellogenesis, beginning at stage 8 . This effect is common to mutants unable to produce large amount of proteins, having reduced levels of either ribosomal proteins, 18S, 28S, or 5S rRNAs. The genomic region adjacent to the PZ transposon was cloned from the mfl 1 stock by plasmid rescue and used to isolate the sequences encompassing the PZ insertion site. Genomic probes spanning a region of ∼4 kb surrounding PZ insertion identified on Northern blots of poly(A) + RNA two main transcripts of 1.8 and 2.0 kb in length, whose expression was affected in each mfl mutant line (see next section). While the 1.8-kb species was constitutively expressed throughout the life cycle, the 2.0-kb RNA was specifically found in adult female and embryonic RNA preparations, in which a further transcript of ∼4.0 kb was also occasionally detected . However, no cDNA representative of this mRNA subform was isolated after extensive screening of an adult female cDNA library, so that it remains unclear whether it actually derives from the mfl gene. In contrast, several cDNAs representative of the 1.8 and 2.0 kb were isolated from adult female and larval libraries. The longest cDNAs of each class, respectively, of 1,833 and 2,034 bp, including the poly(A) tail, represented almost full-length transcripts and allowed us to define the mfl gene structure by Southern blot hybridization and alignment with nucleotide sequence of the genomic region. In each mfl mutant line, a copy of P was inserted at the 5′ common end of 1.8- and 2.0-kb transcription units: in mfl 06 , the insertion site was mapped 18 nt upstream from the 5′ end of the longest cDNAs obtained, in mfl 1 18 nt downstream, within the 5′ leader sequence, while in mfl 05 the insertion occurred within the first intron of the gene . The 1.8- and 2.0-kb mfl mRNA subforms share a common coding region and differ from each other only at their alternatively spliced 3′ untranslated region, where two additional exons (7 and 8) are specifically included in the 2.0-kb mRNA. When used on northern blots, a probe derived from these two exons (probe 2, depicted below the genomic map) detects exclusively the 2.0-kb subform, specifically present in embryos and adult female RNAs . Hybridization of this probe to whole mount preparations of wild-type ovaries reveals that the female transcript accumulates in germ line cells from the early germarial till last oogenesis stages . We then followed the accumulation profile of both mfl mRNAs during embryogenesis by developmental northern blot analysis of carefully synchronized embryos. As depicted in Fig. 3 e, both mfl mRNAs are detected in very early, 0–2 h embryos. However, while the zygotic 1.8-kb mRNA persists at later stages, the level of female transcript drops subsequently, and becomes very low in 4–6 h embryos. This developmental pattern is very similar to that of other stable maternally supplied RNAs, which persist from early stages up to gastrulation. The mfl open reading frame (ORF), identically present in both mRNA subforms, encodes a predicted protein of 508 amino acids with a calculated molecular mass of 56 kD. Database searches revealed that this protein belongs to the Cbf5p/NAP57/dyskerin family . The MFL polypeptide shows a significant degree of conservation to other members of the family, particularly with the two very similar rat and human proteins (66% identity, 79% similarity to human dyskerin). The conservation increases remarkably within several specific domains, strongly underlining that their function has been preserved during evolution. As depicted in Fig. 3 f, total identity exists among Drosophila and human proteins within the two TruB motifs which have homology with bacterial and yeast tRNA pseudouridine synthases . A repeated hydrophobic domain, possibly involved in the nucleo-cytoplasmatic shuttling postulated for the rat protein is also highly conserved. This domain is immediately followed by a block of >20 amino acids having a central tyr that is identical in Drosophila , rat, and human proteins. Although no function has been suggested so far for this domain, its conservation suggests that it might play a relevant role in protein activity. Within the tyr domain, we noticed a RX-x(2,3)-DE-x(2,3)-Y central core motif highly conserved among uracil-DNA glycosylases from different organisms as part of the rigid uracil-binding pocket (Up) present in these repair enzymes. Within the pocket, the tyrosine residue has been shown to be directly involved in uracil recognition . By analogy, it is reasonable to suggest that the highly conserved tyr motif might play a similar role in uracil recognition. A highly charged lysin-rich COOH-terminal region containing a nuclear localization signal is found in MFL, as in NAP57 and dyskerin, and the NH 2 -terminal nuclear localization signal observed in rat and human proteins is also preserved. Finally, it is interesting to note that all five missense mutations thus far identified in DKC patients fall into regions that are conserved between the human and the Drosophila gene . When mfl mutants were checked for gene expression, we found that they were all characterized by reduced levels of mfl mRNAs. While the viable, hypomorphic mfl 1 allele showed only a modest reduction, mfl expression was strongly disrupted in mfl 05 and mfl 06 , the two alleles causing larval lethality. MFL protein accumulation strictly paralleled the level of mfl mRNAs, so that it was strongly reduced in mfl 06 and nearly null in mfl 05 . Remarkably, the developmental time at which lethality is achieved in these two mutants correlates well with MFL level since, as mentioned above, mfl 05 homozygotes die mainly as first instar larvae, while mfl 06 animals as second or early third-instar larvae . Note that, considering the timing of persistence of maternal rRNA , mortality at the first instar is that which may be expected for mutations causing severe loss of function of a gene essential for rRNA processing. Taken together, all these data indicated that MFL level may be critical for Drosophila viability. We then attempted to rescue mfl lethal phenotype by ectopically expressing MFL from the heat-inducible hsp70 promoter. mfl 05 and mfl 06 transgenic animals were then obtained and daily treated at 37°C for 30 min. These heat-shock conditions usually produce amounts of the ectopically expressed protein that largely exceed the wild-type level. However, in our experiments they produced a MFL level just comparable to that present in wild-type flies, even though the induced protein remains quite stable from 6 h to as long as 24 h from the heat-shock pulse . Nevertheless, the level of induced MFL is sufficient to allow mfl 05 and mfl 06 transformed animals to developed synchronously with their wild-type siblings and to show a normal increase in their size. Moreover, 30% of the mfl 05 and 80% of the mfl 06 transgenic animals develop up to the pupal stage, although these transgenic pupae all failed to eclose adult flies . A possible explanation for this partial rescue of the mfl mortality is that the level of ectopically expressed MFL may be inadequate with respect to the rate of protein synthesis required in specific cell types during metamorphosis. An alternative possibility is suggested by the observation that, in yeast, Cbf5p is required for the stability of other components of the H/ACA class of RNPs, such as Gar1p and box H/ACA snoRNAs . It is thus possible that the MFL level reached under heat-shock conditions may not be constant and this affects the stability of other essential RNP components. However, since no member of the H/ACA class of RNPs has yet been described in Drosophila , this hypothesis cannot be tested at present. Given the similarity existing between the mfl phenotype and that caused by mutations affecting the synthesis of ribosomal components, we checked the role of the gene on rRNA processing. Electrophoresis of larval rRNA labeled by [ 3 H]uridine incorporation showed that pre-rRNA processing is inefficient in mfl mutants. In fact, with respect to wild-type flies, increased levels of the pre-rRNA and 28S rRNA and reduced amounts of the 18S, 28Sa, and 28Sb mature species were observed . MFL over-expression in mfl transgenic flies is sufficient to reduce rRNA precursor accumulation and to increase the level of the newly synthesized 18S and 28S species . Northern blot analysis with three different probes derived from the rDNA internal transcribed spacer (ITS) led us to define in greater detail the abnormal rRNA processing occurring in mfl mutants. In Drosophila the rRNA primary transcript (pre-rRNA) undergoes two alternative types of initial cleavages . The most predominant type occurs in the external transcribed spacer, at site 1, and generates the large type a molecule, from which both 18S and 28S are derived . An alternative cleavage occurs within ITS, at site 3, generating the intermediate d and b forms which are, respectively, 18S and 28S rRNA precursors . Hybridization to a probe derived from the ITS 5′ end (probe I) revealed that the accumulation of the pre-rRNA observed in mfl mutants is accompanied by a reduction of the type a precursor and by an increase of the d form; both effects become more evident with progression of the larval development . Thus, mfl mutations specifically affect site 1 cleavage, inhibiting the formation of type a molecules and the processing of the d intermediate. With pathway blocked, pre-rRNA processing proceeds mainly through pathway β, generating equimolar amounts of d and b intermediate molecules. This is confirmed by hybridization to probe II, which shows that, while in wild-type animals the amount of form b largely exceeds that of d (as expected, being that the b molecule is actively produced by both α and β pathways), in mfl mutants these two forms are detected in similar amounts . However, since the processing of form d is inhibited, this species accumulates progressively along larval development . Conversely, hybridization to probe III indicated that mfl genetic depletion does not impair site 4 cleavage of type b molecule, since the amount of form c observed in the mutants exceeds even that of the control . We concluded that form c is generated properly, but its further processing is inhibited by mfl mutations. In mfl transgenic flies, MFL over-expression leads to a reversal of all of the effects observed, although the efficiency of pre-rRNA processing is not fully restored. In heat-shocked transformed animals, in fact, MFL expression causes a decrease of pre-rRNA accumulation and an increase in the production of the type a molecule . Processing of the type a precursor also occurs properly, since, as depicted in Fig. 5 d, these larvae show an excess in form b versus form d, although the amount of the b molecule does not reach that observed in wild-type animals. Finally, the amount of form c appears reduced after the heat-shock , indicating that its processing is at least partially restored. In yeast, lack of Cbf5 gene activity affects not only rRNA processing, but also rRNA pseudouridylation. Thus, we checked the level of modification in wild-type and mfl mutants at several 28S and 18S Ψ specific sites. With this aim, we used oligonucleotide primers complementary to selected 28S or 18S regions to perform primer extension analyses on CMC-treated Drosophila rRNA. CMC blocks reverse transcription, resulting in a gel band terminating in one residue 3′ of the Ψ site . In planning these experiments, we took advantage of the location of Drosophila 28S rRNA pseudouridines recently reported by Ofengand and Bakin . Instead, none of the 18S Ψ sites checked in our experiments was previously known. In spite of the persistence of maternal rRNA, pseudouridylation appears reduced in mfl 05 larvae at several 28S sites, such as the U2442, U2444, and U2499 residues . Similar reduction was observed at various 18S rRNA sites, such as U830/U831, U840, U841, and U885 , indicating that, as Cbf5 , mfl is required for efficient rRNA pseudouridylation. An unexpected feature of the mfl gene structure was revealed by the finding that a small RNA species, ∼0.1 kb in length, hybridized specifically with the genomic sequences of the fourth mfl intron, while it was not detected by any cDNA probe. This small RNA was detected in total RNA preparations from all developmental stages and was specifically enriched in the poly(A) − RNA fraction. The length of the small RNA species was accurately determined on denaturing 6% polyacrylamide gels and its 5′ end precisely mapped by primer extension analysis of total larval RNA using two different oligonucleotides . These experiments pointed out that this transcript was ∼140 nt long and derived from position +37 to about +176 of the 235-nt-long fourth mfl intron . Since a large number of small nucleolar RNAs are intron encoded , we checked for the presence of conserved snoRNA elements within the 0.14-kb RNA sequence. Two H boxes (consensus ANANNA) and a 3′ terminal ACA element were found ; in addition, the predicted secondary structure of the mfl intron-encoded RNA conformed well to the hairpin-hinge-hairpin-tail architecture common to most yeast and vertebrate box H/ACA snoRNAs . Two short regions of complementarity between the mfl intron encoded RNA and Drosophila 18S rRNA were also found . As noticed by Ganot et al. , short regions of pairing with rRNA flank the site of pseudouridylation, allowing the positioning of the residue to be isomerized at the base of the stem, at the first unpaired position before the 3′ snoRNA helical segment. The pseudouridine selected is found to be separated from the H or ACA box by 14 or, in a few cases, by 15 nucleotides. On the basis of these observations, the rRNA pairing properties of the mfl intron-encoded RNA predicted it may direct pseudouridylation of Drosophila 18S rRNA at position U1820 . Primer extension analysis on CMC-treated Drosophila rRNA shows that the potentially selected residue is actually pseudouridylated . The selected U1820 residue is equivalent to U1698 of human 18S rRNA, whose pseudouridylation has recently been related to the U70 snoRNA . As for U1698 in human rRNA, the Drosophila U1820 residue is the first of three consecutive uridines, all of which are pseudouridylated . In yeast, genetic depletion of most of the box H/ACA snoRNAs has been reported to inhibit pseudouridylation of the specifically selected sites . When we checked modification of the U1820 residue in rRNA preparations obtained from mfl 05 first instar larvae, we found that pseudouridylation was reduced not only at U1820, but also at U1821 and U1822 residues . This result may be explained by the widespread inhibition of rRNA pseudouridylation observed in mfl mutants. Further experiments are thus required to define the specific functional role, if any, played by the mfl intron-encoded RNA. Finally, we checked the localization of the mfl intron encoded RNA by in situ hybridization experiments to whole mount ovary preparations. This analysis showed that a 0.14-kb RNA-specific antisense probe exclusively labeled the nucleoli as it occurs in each tested embryonic or larval tissue (not shown). Specific nucleolar localization may also be observed for MFL , whose ubiquitous expression resulted from both immunolocalization data and histochemical staining of lacZ activity in mfl 1 flies (data not shown). In ovarian tissue preparations we noticed that the protein occasionally diffuses into the cytoplasm in several patches of follicle cells. As judged by the presence of well defined, round-shaped nuclei having morphologically well distinguishable nucleoli , these cells should not be in or around mitosis. Moreover, cytoplasmic diffusion can be observed also after stage 10b of oogenesis, when follicular cells endocycles are reported to be terminated . It is thus plausible that occasional MFL cytoplasmic localization may be related to ability to carry out nucleolus-cytoplasmic shuttling, as proposed for NAP57 in rat cells . Taken together, the experiments reported indicate that mfl hosts, in its fourth intron, a box H/ACA snoRNA gene, the first member of this class to be identified so far in Drosophila . We have called this gene snoH1 and suggest that it is functionally equivalent to the human U70 snoRNA gene. We reported the cloning of the D . melanogaster mfl gene and established that it encodes an ubiquitous nucleolar protein essential for Drosophila viability and female fertility. Our data also showed that mfl is closely related to the other members of the Cbf5 family so far characterized from higher eukaryotes, the rat Nap57 and the human gene responsible for the X-linked dyskeratosis congenita disease. As cogently predicted , flies carrying mutations in the Drosophila DKC1 orthologue show a pleiotropic phenotype very similar to that caused by mutations that affect the synthesis of ribosomal RNA. In fact, we found that mfl loss-of-function mutations impair rRNA processing and lead to accumulation of rRNA precursors. Although these effects are very similar to those caused by Cbf5 genetic depletion, yeast mutations preferentially affect the production of mature 18S rRNA , while mfl mutations cause similar reduction of 18S and 28S rRNA species. It would be of interest to know whether this is due to a distinctive feature of Drosophila rRNA processing pathways, or whether it reflects a general property of rRNA processing in higher eukaryotes. In addition to affecting rRNA maturation, mfl loss-of-function causes reduced levels of pseudouridylation at several 28S and 18S Ψ sites, suggesting that gene activity might be required for fully efficient rRNA pseudouridylation. Again, these results are reminiscent of those obtained in yeast , and outline the existence of a link between rRNA processing and rRNA pseudouridylation in eukaryotes. By mapping the protein domains conserved among members of the Cbf5p family and investigating the definition of their functional roles, significant information should be generated about the functional role played by rRNA pseudouridylation, which still remains elusive. Although pseudouridylation of eukaryotic rRNAs occurs predominantly on the primary rRNA transcripts before nucleolytic processing, this type of modification is not required for efficient processing of 25S yeast rRNA . It has been suggested that pseudouridylation can contribute to rRNA folding, rRNPs assembly, and ribosomal subunit assembly . Other hypotheses, such as subtle enhancing of ribosomal functions or influencing fidelity of codon recognition, have also been proposed . An additional role that could be suggested for MFL is based on the observation that it can occasionally diffuse within the cytoplasm. As previously suggested for NAP57 in rat cells, it is tempting to speculate that this may possibly reflect the ability of MFL to structure and export pre-ribosomal RNP particles into the cytoplasm. If confirmed, this would strongly support the view that members of this family are multifunctional proteins involved in different aspects of ribosome biogenesis. It is possible that these proteins may constitute essential components of a single multifunctional complex or, alternatively, they represent common components of structurally and functionally different RNP particles. The definition of the functional interactions required to carry out such a variety of functions will help to clarify this point. Remarkably, the identification and the characterization of mutations disrupting mfl gene expression has led to establishing the first animal model system for the study of the X-linked dyskeratosis congenita human disease. Some of the results reported here may immediately provide useful information for the comprehension of the molecular basis of the DKC disease. A first relevant point concerns the observation that none of the mfl mutations so far isolated disrupts the gene coding region. Thus, each Drosophila mutant line has certainly quantitative and not qualitative alterations of the gene product which causes the pleiotropic abnormalities observed. The level of MFL protein was found to be critical, and a simple dose-effect rule may be derived: when the protein level is below a crucial threshold, mortality ensues. Instead, while the protein level is lowered but still stands above a critical threshold, the viable, hypomorphic mfl 1 phenotype is reached. By analogy, it can be suggested that in man the level of dyskerin activity may be one of the critical parameters able to trigger the DKC disease. The finding that DKC mutations mapped so far all affect the dyskerin coding region is in only apparent contrast with that found in Drosophila . In fact, it is reasonable to suppose that, as observed in Drosophila , total or severe loss-of-function mutations should not be compatible with life. Mutations recovered in patients might be those causing partial loss-of-function, so that the level of dyskerin activity is still compatible with survival. Accordingly, DKC patients might carry hypomorphic mutations, the human counterparts of the viable mfl 1 phenotype. Whether these hypomorphic phenotypes are simply a consequence of the inadequate mature rRNA level or are, at least partially, caused by abnormal accumulation of intermediate rRNA species is an important point which deserves further investigation. A further issue concerns the observation that, although MFL and dyskerin are ubiquitous proteins, phenotypic abnormalities are, in Drosophila as in man, restricted to only certain tissues. Since the gene product is presumed to be critically important for protein synthesis in every cell of the body, the finding that abnormalities are developed only by selected cell types is quite surprising. However, if it is accepted that the level of protein activity may be a critical parameter, then it is reasonable to suppose that the amount of properly processed rRNA may be sufficient in cells having a slow growth rate, while in highly proliferating tissues or in cells sustaining a high rate of protein synthesis this would not the case, and degenerative cell defects could progressively be accumulated. Interestingly, inhibition of protein synthesis is known to be one of the stimuli capable of inducing apoptotic cell death, probably by decreasing the levels of essential proteins or by inhibiting the synthesis of proteins that normally suppress the spontaneous activation of apoptosis . In mfl 1 ovary, one of the Drosophila tissues where morphological abnormalities can be observed, we found that degeneration is specifically accompanied by apoptotic cell death. This observation might also suggest a role for apoptosis in the progressive clinical manifestations of the DKC disease. Finally, we showed that mfl gene organization is intriguing, and leads to the identification of the first member of the box H/ACA class of snoRNAs described so far in Drosophila . As in the case of the snoH1 gene described here, most of the snoRNAs are intron encoded, and snoRNA host genes often encode proteins involved in translation or ribosome biogenesis . These intron-encoded snoRNAs are cotranscribed with their host pre-mRNA and their accumulation is splicing-dependent, since they are released from the excised intron by exonucleolytic processing. Our observation that snoH1 RNA and mfl mRNAs levels are reduced in parallel in each mfl mutant line strongly suggests that snoH1 RNA processing is linked to the splicing of the mfl primary transcript. This feature, which allows coordinated regulation of the host protein and the intron encoded snoRNA, may hinder a precise definition of the specific functional role played by each product. With regard to snoH1 , it cannot be excluded, in principle, that it may be required for Drosophila viability and that its depletion might contribute to the generation of mfl phenotype. However, we have observed that snoH1 has little, if any, effect on mfl phenotypic rescue when over-expressed in mfl transgenic flies, either in the presence or in the absence of MFL overexpression (Giordano, E., and M. Furia, unpublished data). This is not surprising, given that all box H/ACA snoRNAs found in yeast, with the exception of snR30 and snR10 , are dispensable for viability. It will now be interesting to determine whether this type of gene organization is restricted to mfl or is shared by other members of this conserved gene family. | Study | biomedical | en | 0.999998 |
10087259 | Normal rat kidney cells were cultured at 37°C in DMEM ( Sigma Chemical Co. ) supplemented with 8% fetal calf serum. For specimen preparation, cells were grown to ∼80% confluency on Formvar-coated, carbon-stabilized, glow-discharged 100-mesh gold EM grids. Cells were maintained at 37°C until within 15 s of the freezing step. Grids with cells attached were picked up with self-closing forceps and rinsed briefly (∼10 s) in DMEM containing 3% 70-kD Ficoll ( Sigma Chemical Co. ), which serves as an extracellular cryoprotectant. The addition of Ficoll, including its contaminating salt, increased the osmolarity of the medium by <3%. The forceps were then attached to the piston of a plunge-freezing apparatus. All excess fluid was carefully removed from the grid with filter paper (#1; Whatman Inc.), and the piston was released to plunge the grid rapidly into a pool of liquid ethane chilled to −174°C by a surrounding bath of liquid nitrogen. The rate at which samples are cooled by this method is ∼104°C/s . After plunging, grids were quickly transferred to liquid nitrogen and prepared for freeze substitution. For freeze substitution, the samples were transferred first to cryostorage tubes (Nalge-Nunc International) containing 1% glutaraldehyde and 0.1% tannic acid in acetone and maintained at −90°C for 2 d. They were then allowed to warm slowly to −50°C over the course of ∼6 h, at which point the substitution solution was replaced with a precooled (−50°C) solution of 2% OsO 4 and 0.01% uranyl acetate in acetone. Samples were then allowed to warm from −50° to 4°C over 24 h. The samples were rinsed three times in acetone, infiltrated with increasing concentrations of Epon-Araldite resin, and flat-embedded between two Teflon-coated glass microscope slides (Miller-Stephenson). The resin was polymerized at 60°C for 2 d. Embedded samples were observed by phase-contrast light microscopy and areas of apparently well-preserved cells were excised from the plastic “wafer” and remounted with epoxy glue onto plastic stubs in an orientation suitable for cross-sectioning. Thin (30–40-nm) sections were cut on a UltraCut-UCT ultramicrotome (Leica Inc.), transferred to Formvar-coated copper-rhodium slot grids, stained with 2% aqueous uranyl acetate and Reynold's lead citrate, and observed on a JEM-100CX transmission EM (JEOL U.S.A. Inc.) to determine specimen quality and to select suitable samples. Blocks that yielded cells with distinct regions of well-preserved Golgi were returned to the microtome where ribbons of 6–12 serial 250-nm sections were cut and transferred to slot grids. Sections were stained for 15 min with 3% uranyl acetate in 70% MeOH, and then 3 min with Reynold's lead citrate. After staining, 10 nm colloidal gold particles were added to both sides of the grid to serve as fiducial markers for aligning the series of tilted images. A second layer of Formvar was cast onto the section side of the grid, and both sides of the grid were carbon coated to enhance stability. Procedures for image acquisition and dual-axis tomography were as described previously . In short, grids were placed in a stage capable of both high tilt and specimen rotation in the plane of the grid. They were observed in a JEM-1000 HVEM (JEOL U.S.A. Inc.) operating at 1 MeV. Once a suitable Golgi region was selected, the sample was tilted from +60° to −60° at 1.5° intervals, and 80 images at 15,000× were collected on film (23D56; Agfa-Gevaert N.V.). The grid was then rotated by 90°, and a similar series was taken. An area corresponding to 3.84 × 1.86 μm was digitized at a pixel size of 2.35 nm. Images were aligned and a tomogram was computed from each tilt series. To merge the two single-axis tomograms into one, they were registered to each other with a warping procedure, rather than a single linear transformation , because nonlinear distortions between the two tomograms were evident over such a large volume. The above procedure was followed for each of the four serial sections. It is difficult to specify the resolution of our reconstruction with certainty. Standard formulas give a resolution of 7.8 nm for each of our single-axis tomograms in the x direction (perpendicular to the tilt axis). For several reasons (e.g., the appearance of the unit membrane bilayer in some areas), we estimate that the resolution in the x–y plane for our dual-axis tomograms is ∼6 nm. Because of the limited range of tilt angles, resolution is 1.3× worse in the z direction , or ∼8 nm. The tomograms from adjacent 250-nm sections were aligned with each other by examining 2.4-nm slices extracted from the tomograms parallel to the plane of each section; the bottom-most slice from the first section was aligned to the top-most slice from the next. This method was similar to that of Soto et al. , except that we aligned the pair of slices with a general linear transformation, which incorporates an overall size change and stretch along one axis. Such a transformation was necessary to obtain a good match across the section boundaries, which in turn was important for being able to track features across a section boundary. The parameters of the transformation were adjusted manually to give the best apparent fit, as judged by viewing the two slices in rapid alternation . The linear transformation gave an excellent alignment of the slices across two of the boundaries and an adequate match across the third, with displacements of two to three pixels in some regions of the reconstruction. The alignment process was complicated by an evident loss of material between sections. The amount of loss was assessed by examining the change in image features across a section boundary; vesicles cut by the sectioning process were particularly useful. Approximately 15–25 nm of material appeared not to be present in the tomogram. On rare occasions, these gaps led to ambiguity in establishing connectivity between vesicular or tubular elements. Once the alignment parameters were determined, aligned regions (1,632 × 440 × ∼65 pixels) were extracted from each of the four sections and stacked into a single volume 257 pixels thick. The model presented here was constructed from this volume. The thickness of stained material within each section of the tomogram was only 150 nm, partly due to the loss of material just described, but mostly due to the collapse in section thickness that rapidly follows the initial observation of plastic sections . A 2.4-nm slice from the tomogram parallel to the plane of section thus corresponds to ∼4 nm of unthinned material. Before viewing and analysis, the z dimension of our model was stretched by a factor of 1.65 to bring the total z extent of the model to 1.0 μm (four sections of 250-nm each). The tomographic reconstruction was interpreted and modeled using Silicon Graphics computers running the IMOD software . Each compartment in the reconstructed Golgi region was considered a distinct “object,” and a different color was assigned to each object. The portion of each object that was visible in one tomographic slice was traced as a “contour” overlaid on the image. Objects were modeled one at a time. Before modeling an object, the operator studied it throughout its entire volume to make an initial assessment of its connectivity. Modeling began at a slice within the data set where the object was most distinct; it was then traced through adjacent slices in both directions to assure that all compartments associated with that object were included in the model. This often involved backtracking to include compartments that in some slices were separated from the main body of the object, but in others were obviously attached to it. If a coat was present on a budding profile, its position was indicated by stippling. The resolution of the tomogram allowed for most objects to be followed and modeled with complete confidence. However, some tubules in the noncompact region, budding profiles, and the regions at the boundaries of physical sections, posed a challenge. These regions were given particular attention by using the “slicer” tool in IMOD that allows the operator to view image data from any angle. Free vesicles were modeled in one of two ways. Vesicles located within openings in the Golgi stack were modeled as described above. Their small radius, combined with the slightly reduced resolution of our tomograms along the beam axis, led to a modeling artifact. Each of these vesicles appears slightly extended parallel to the beam axis. Free vesicles located around the periphery of the stack were modeled by placing a simple sphere at that location. Vesicles with different coat structures were marked by spheres of differing size and color. After its completion, the model was smoothed by fitting local polynomials to the surfaces and replacing each point with a corresponding point from the fitted surface. This smoothing compensated, in part, for the difficulty in drawing contours that shifted smoothly in position from one plane to the next. A mesh of triangles was then computed to define the surface of each object , so that objects could be viewed with standard lighting techniques. Surface areas in the model were computed from the triangular meshes, and internal volumes were determined from the contour information, after adjusting for the thickness of the membrane inside the contour. The sizes of openings in the cisternae were measured with a program devised for this purpose. The operator first marked the top and bottom of each opening with model points. The program then found the projected area of the opening from the angle of view that gave the greatest area. For each opening, the diameter of a circle with the same area was computed and used for analysis. Accurate 3-D modeling of cellular fine structure requires both excellent preservation of the biological sample and a method for precisely tracking objects in space. Here we have used a combination of cryofixation and dual-axis, HVEM tomography to model the Golgi complex with a 3-D resolution of ∼7 nm. The quality of the structural preservation provided by fast freezing and freeze substitution, as well as the resolution of the tomographic “slices” used in this study, are demonstrated in Fig. 1 a. The Golgi cisternae of this cultured NRK cell are smooth and comparatively straight. The spacings between adjacent cisternae are remarkably uniform, and all of the surrounding cytoplasm appears to be well preserved, including cytoskeletal elements such as microtubules . The tomographic slices used in this study are only 4-nm thick, so they offer a significant advantage over typical serial “thin sections” (∼60 nm) for tracking convoluted membranes in 3-D. This advantage is evidenced by the very slight but detectable change in the microtubule from Fig. 1 , a to b, which is the adjacent tomographic slice. Nearly all the budding profiles on Golgi cisternae displayed a coat of some kind. The fixation and staining procedures employed in this study have enabled us to distinguish membrane bilayers , noncoated vesicles (b, c, and c′), nonclathrin–coated buds and vesicles (d, e, and e′), and clathrin coats (f, f′, g, and g′). Although clathrin and nonclathrin coats can be distinguished from uncoated vesicles, we cannot distinguish COPs from “lacelike” coats . Our description of coats is therefore limited to clathrin and nonclathrin. The reconstruction shown here is derived from four serial 250-nm sections that yielded 257 tomographic slices, like the ones shown in Fig. 1 , a and b (slices 175 and 176). All of these slices were used to model the shapes and positions of seven Golgi cisternae and the nearby ER, vesicles, and tubules. The boundary of each Golgi element was traced to represent its membrane in each tomographic slice, as described in Materials and Methods. Different colors were used to represent each cisterna and its associated structures . The quality of the individual images and the thinness of the slices made for essentially no ambiguity in the connectivity of the different Golgi compartments. This factor allowed us to look carefully for places where adjacent cisternae might fuse or connect, but we found none. The volume selected for this reconstruction (∼1 × 1 × 4 μm) included two compact Golgi regions separated by a noncompact region (NCR); together, these constituted ∼5% of the cell's entire Golgi complex, based on estimates from low magnification EM (not shown). When the resulting model is displayed in its entirety, it is so complex that single views are not as informative as one would like . Although the ER at the top and bottom and the well ordered cisternae of the compact regions are clear, the less ordered regions of the model are more difficult to visualize. The fine details within the model are obscured by all the features that are superimposed. We therefore present our results by disassembling the model in various ways. One of the most informative views of the model is shown in Fig. 3 (top), where the individual layers, including the Golgi cisternae C1–C7, are displayed as separate objects, each viewed from its cis-Golgi side. The cis-trans polarity of this Golgi region was deduced from the orientation of the cis-Golgi wells and the fact that only cisterna C7 displayed buds coated with clathrin. Clathrin has been shown to be associated only with the trans side of the Golgi . Other features of the model are highlighted below . On the cis side of the reconstructed Golgi is a layer of ER . This ER displays typical tubular and cisternal domains but no budding profiles. The lack of budding suggests that the cis ER reconstructed here was not functioning as an “exit site” at the time it was frozen. The paucity of cis ER in this model reflects the limited volume digitized. Between the cis ER and the stacks of Golgi cisternae, there is a layer of branched tubular structures and flattened sacs . Most of these 44 discrete elements are concentrated over the compact regions of the cis-most cisterna (C1), suggesting a specific association. Based on its position and morphology, we interpret this layer as part of the ER-Golgi intermediate compartment (ERGIC). None of the ERGIC is, however, closely adherent to the surface of C1 . Some of the ERGIC elements exhibit structures that resemble budding or fusing vesicles, but none appears coated. There is no continuity between the ERGIC elements and either the cis ER, C1, or any of the other Golgi cisternae. The ERGIC is structurally different from all of the Golgi cisternae, and indeed an examination of the yellow contours in Fig. 1 b suggests that if only 2-D information were available, one might not identify the ERGIC as a compartment with a distinct structural character, but rather as a collection of polymorphic vesicles. The ERGIC region also contains many spherical vesicles, as shown in Fig. 3 (bottom). A single microtubule enters the ERGIC region from the edge of the sample and runs approximately parallel to the plane of the Golgi cisternae . Each of the compact regions in this Golgi model consists of seven stacked cisternae. There is also a partial cisterna (C6′), which is found only on the right side of the right stack . Bridges across the NCR connect the two parts of three cisternae (C1, C3, and C6), while C7 nearly covers the NCR, blocking most of its access to the trans side of the Golgi. Bridges were not detected between the parts of C2, C4, and C5, but these cisternae may be connected outside the reconstructed volume. There are no lateral, vertical, or branched connections between cisternae at different levels in the stacks. Although each cisterna is distinct, these structures possess many features in common. All cisternae are interrupted by openings of three different size classes (discussed below). All display buds, which are usually located at their margins or at the edges of holes . On C2 and C6 budding structures are also found at the distal ends of tubules that extend from the margins of the cisternae. C6 and C7 also show one bud each forming from their flattened surfaces . Most of the buds associated with the Golgi cisternae appear coated. The few that lack coats invariably also lack a necklike constriction, suggesting that they are images of fusing vesicles. When the cisternae are aligned, their larger holes line up to form “wells.” In contrast, the smaller openings, called fenestrae, are not in register from one cisterna to the next. Free vesicles and budding profiles attached to the cisternal edges that delineate the wells fill much of the well volume . Note that the nonspherical appearance of the vesicles is probably due to the stretching artifact described in Materials and Methods. There is also a tapering tubule, depicted with Fig. 3 , C5, that extends from the edge of the model into one of the wells . Cis-Golgi wells are those that are open to the cis side of the stack but are closed at the trans side . Fig. 3 contains two cis-Golgi wells in the left compact region and one in the right. These wells penetrate through different numbers of cisternae: C1–C3 and C1–C6, respectively. Another set of aligned holes in the right stack has no opening to the cis face; its access to the cytosol is through an opening on the side of the stack . It too contains profiles of coated budding vesicles. Yet another set of aligned openings forms a “trans-well,” but this well lacks both budding and free vesicles . Tubules with budding tips project from the margins of both cis- and trans-side cisternae; they extend outwards, roughly perpendicular to the planes of the cisternae . Tubular extensions from multiple trans cisternae have been used as a defining characteristic of the trans-Golgi network , but tubules emanating from cis cisternae have been reported less frequently . Both of the tubules that emanate from C2 end in the ERGIC region with a coated bud . Cisternae C5, C6, and C7 all extend tubules into the region that lies trans from C7 . The C6 cisterna is unique in that it extends one tubule in the cis direction as well as one in the trans . The cis-oriented tubule on the left passes all five of the preceding cisternae and finally ends in the ERGIC region with a coated bud. The 3-D resolution of our model has enabled us to define the contents of an NCR better than has previously been possible. Most workers show this region filled with vesicles that lie between slender bridges connecting cisternae of adjacent stacks . Fig. 5 b illustrates at higher magnification a projection of the elements in the NCR that are not connected to cisternae. The region contains numerous vesicles, which are colored white, in addition to larger polymorphic elements. Fig. 6 shows most of the elements of the NCR that are attached to cisternae. These three sets of bridges and tubules are complex and, like some of the free elements, polymorphic. The origin of the free polymorphic elements is a fascinating question. Several of them resemble the tubular and flattened elements of the ERGIC . Others are more rounded and have a smaller surface-to-volume ratio than any elements of the ERGIC . Some disconnected elements of both colors resemble the bridging and tubular structures that are connected to cisternae in the NCR . Fig. 6 depicts a total of 394 vesicles, including the free vesicles in the wells, the NCR, and the peripheral regions of the reconstructed Golgi. Of these, only eight are clathrin coated. The remaining population is nearly equally divided between non–clathrin-coated (190) and noncoated vesicles (196). The total surface area of the vesicle membranes is 4.9 μm 2 , comparable with the surface area of one cisterna of our model, and their total volume is 0.025 μm 3 , similar to the volume of C5 . A majority of the non–clathrin-coated vesicles is localized around the cis half of the stacks, whereas more of the noncoated vesicles are around the trans half . This figure also shows that the distribution of vesicles at the trans side of the model is highly nonuniform. The trans ER shown in Fig. 5 , d and e, flattens and closely abuts the trans faces of both C7 and C6. These appositions appear as tight as those between adjacent Golgi cisternae. In addition, the trans ER is wrapped around parts of C7 . Ribosomes are associated with the trans ER on only the side that is distal to the Golgi. We have also observed such interactions in both PtK and CHO cells after fast freezing/freeze substitution (data not shown). Our observations suggest that this relationship is characteristic of mammalian Golgi. The trans ER lacks buds, suggesting that there is no vesicular transport between this compartment and the Golgi. The partial cisterna located between C6 and C7 has the same general morphology as the trans ER, except that it is free of ribosomes, it lacks coated buds, and displays an intimate association with both trans-Golgi cisternae. The descriptions presented above are all based on one model of a portion of the Golgi ribbon from one cell. We think, however, that they are more representative of the structure of NRK Golgi than this small sample might suggest. While developing our methods for specimen preparation, we prepared ∼40 sets of samples by cryofixation and freeze substitution, and then evaluated them by serial thin sectioning and in some cases by HVEM tomography. All major observations described here (lack of connectivity between nonequivalent cisternae, association of clathrin with only the trans-most cisterna, the existence of tubules projecting from multiple cisternae, and the close association of trans ER with multiple trans-Golgi cisternae) have been reproduced in two or more cells and sometimes in multiple cell types. The labor of detailed modeling (6 × 10 6 data points recorded here) has precluded equivalent documentation of a large number of cells. The gallery of images in Fig. 3 demonstrates the extent of structural variability among the modeled Golgi cisternae. We have quantified several structural parameters of the individual cisternae, some of which can be used to distinguish them. The surface areas of the reconstructed cisternae are remarkably similar, varying by <15% . In contrast, the volumes of the cisternae differ by as much as 50% , with C1 and C7 having the greatest volumes, C2 and C6 being ∼20% less, and C3–5 having the smallest volumes. The ERGIC elements collectively have a markedly smaller surface area and volume than any of the Golgi cisternae. The cisternae differ significantly in the size, density, and distributions of the openings that penetrate them . The distribution of opening sizes for all cisternae shows three modes, so the openings can be subdivided into “fenestrae” (<65 nm), “small holes” (65–100 nm), and “large holes” (>100 nm), that form the wells. The distribution of fenestral sizes changes progressively from cis to trans . Thus, in cryofixed specimens, cisternal openings constitute a quantifiable trait that differs between cisternae. The types and numbers of coated buds are additional quantifiable parameters that distinguish Golgi cisternae. As seen most clearly in Fig. 3 , the coated buds on C7 are all clathrin coated (yellow stippling), whereas those on all other cisternae are exclusively of the nonclathrin type (blue stippling). None of the cisternae exhibits a mixture of clathrin- and non–clathrin-coated buds. The summed volumes of all the buds on a given cisterna are uniformly lower for the C1–C6 cisternae than for C7 (data not shown). The numbers of buds per cisterna vary in the cis-to-trans direction, with the highest numbers associated with C1 and C2, a gradual decrease from C3 to C5, and a leveling off in C6 and C7. The two trans-most cisternae also differ from the preceding cisternae in less quantifiable ways . Whereas the C1–C5 cisternae are all fairly flat and display a typical cisternal architecture that is interrupted only by well-forming holes, C6 is structurally variegated. It has two unusually shaped bridges that connect its parts across the NCR ; it includes a higher proportion of the 65–100-nm class of holes, bulging margins, and both cis- and trans-pointing tubular extensions . The C7 cisterna presents a broad, undulating central region that covers part of the noncompact region. Some of its marginal domains appear inflated. Finally, as mentioned before, only C6 and C7 form close contacts with trans ER. Based on these morphological features, the seven cisternae can be subdivided into two cis (C1 and C2), two medial (C3 ands C4), and two trans (C6 and C7) cisternae, accompanied by one cisterna (C5) that possesses both medial and translike properties. The evidence supporting these assignments rests, in addition to cisternal position, on the quantitative analyses presented above, the presence of tubules emanating from certain cisternae into the ERGIC and TGN regions, and the appositional interactions with trans-ER membranes. Ever since Camillo Golgi first described the “Apparato reticolare interna” , this organelle has been studied intensively . Bursts of new information have often followed improvements in technology. Our use of two advanced structural methods (rapid freezing and dual-axis HVEM tomography) has likewise provided new information about Golgi architecture and novel insights into the relationships between its structure and its function. Many of our findings are summarized in Fig. 9 . Highlights of the data include: detailed qualitative and quantitative information about the architecture of the different types of Golgi cisternae, including their buds, fenestrae, surface areas, and volumes; the description of tubules projecting from multiple cis and trans cisternae; the identification of polymorphic elements in the NCR; a characterization of the membranous elements of the ERGIC; the demonstration and 3-D description of specialized ER that is closely associated with the trans cisternae; and a quantitative analysis of the vesicles seen close to the Golgi stacks. As in most published work, cisternae form the basic units of this Golgi reconstruction. The region modeled contains two compact regions, each composed of seven cisternae that lie on either side of an NCR. Tubular bridges connect the two parts of cisternae C1, C3, and C6, but there are no connections between nonequivalent cisternae. Only the trans-most cisterna, C7, retains its sheet-like architecture over most of the NCR . Photobleaching experiments have shown that Golgi membrane proteins tagged with the green fluorescent protein can diffuse along the entire Golgi ribbon within equivalent cisternae , which suggests that even the cisternae without apparent bridges are connected, perhaps outside the volume reconstructed. Alternatively, bridges may be transient, as suggested by the closeness of the ends of the “fragmented” bridge in C2. Fenestrae (openings with diameters <65 nm) are common to all cisternae. The distributions of their sizes in different cisternae suggest that fenestra geometry is regulated. The functions of fenestrae remain unknown, but we identify three possibilities: they may serve as “spot welds” to control cisternal swelling, they may aid in the formation of buds by increasing the curvature of cisternal margins, and/or they may provide efficient pathways for diffusion from the membrane on one side of a cisterna to the other. We confirm the existence of cis-Golgi wells , and we also identify smaller channels that open to the lateral and trans sides of a compact region. The prevalence of buds and vesicles in cis wells suggests that they are pathways for membrane traffic, possibly in both the anterograde and retrograde directions, though closure on the trans side eliminates the possibility of trans-Golgi exit by this route. The medial cisternae in this reconstruction (C3, C4, and perhaps C5) lack some of the more striking structural elements of the cis and trans cisternae. They are less fenestrated, and their fenestrae are of intermediate size . When viewed together, these cisternae exhibit cis-to-trans gradients in all of the quantified parameters in Fig. 7 , except for surface area. Excepting C5, they lack the striking tubular extensions that are found on cis and trans cisternae. The noncompact region in our model is more complex than previous data have suggested. In addition to the bridging tubules that connect equivalent cisternae from adjacent compact regions, it contains vesicles and polymorphic elements, some of which resemble components of the ERGIC. The extent to which its polymorphic elements contribute to membrane trafficking is, however, unknown. The trans side of the NCR is partially occluded by C7, which may bias its vesicle trafficking toward the cis side. Indeed, it and the cis-Golgi wells may be quantitatively different manifestations of similar Golgi specializations. The structural complexity of the NCR suggests, however, that this region plays additional, yet-to-be characterized roles in Golgi function. All of the Golgi cisternae display budding vesicle profiles. All but 2 of the 92 buds in our model arise from the edges of cisternae, emerging from either their margins or from the rims of their wells. The highest numbers of buds are on the cis cisternae, followed by a decline towards the trans cisternae ; this pattern is consistent with the idea that buds are primarily involved in retrograde transport . However, the presence of large numbers of non–clathrin-coated and noncoated free vesicles surrounding the trans side of the stacks suggests that some of the buds are destined for exit from the Golgi. Future refinements in staining methods should permit further subclassification of coats beyond the nonclathrin and clathrin coats distinguished here. The layer of our model called ERGIC consists of 44 branched, polymorphic membranous elements situated between the cis ER and the cis-most Golgi cisterna . ERGIC was first identified in experiments that used low temperature (15°C) to stall the transport of newly synthesized Semliki Forest virus spike glycoproteins . Markers for this compartment have been identified in two species: p58 in rat and p53 in human cells . Further work has identified “vesicular tubular clusters” (VTCs), which are defined as “export complexes” that form around ER “exit sites” dispersed throughout the cytoplasm . At these sites, newly synthesized secretory and membrane proteins are sorted and packaged into COPII-coated vesicles that subsequently fuse into larger structures from which ER components are recycled via COPI-coated vesicles. The p58/p53 antigen that identifies ERGIC is also a marker for VTCs, so ERGIC and VTCs are considered to be related structures. VTCs have been shown to travel along microtubules and flatten onto the cis face of the Golgi . In our reconstruction, the polymorphic structures of the ERGIC layer resemble elements from VTCs. They also lie adjacent to the cis-most cisterna, though most are not flattened. We suggest that the ERGIC layer of our model is composed of parts of VTCs that have arrived at the Golgi but were frozen before they had flattened on the Golgi stack. The clustering of ERGIC over compact regions of the underlying cisterna suggests an interaction with the cis-most Golgi cisterna and may reflect an intermediate stage in the formation of a new cisterna . Extensive evidence suggests that the cis and trans faces of compact Golgi regions are dynamic and structurally complex. With stereo EM of hyperosmicated specimens, the cis-most cisterna appears as a reticular network with occasional tubular structures extended from it . Tubules have also been observed in vivo projecting from the cis side after antibody injection , and from the trans side, using fluorescent lipids or chimeras containing the green fluorescent protein . These tubules are dynamic, extending and breaking off from the rest of the Golgi. Our images of the cis-Golgi region include tubules , although the ones we have seen are smaller than those visualized by light microscopy. We have not, however, seen the reticulum that is observed in hyperosmicated specimens. This discrepancy might be due to our not having examined enough specimens, but other Golgi regions, visualized in fast-frozen, freeze-substituted cells by HVEM tomography, also lack this feature (data not shown). Experimental conditions that extract or destroy Golgi membrane proteins (e.g., hyperosmication) may destabilize cisternal architecture, enlarging fenestrae, promoting the formation of tubules, and converting fenestrated cisternae into planar tubular networks . We therefore think it likely that reticulation of the cis-Golgi region is induced by preparative procedures. This interpretation suggests a rethinking of the concept of a “cis-Golgi Network” . Our data are consistent with the idea that the two cis-most cisternae, together with their associated tubules, carry out the functions attributed to the CGN, but that there is no cis-Golgi network per se. The trans sides of compact Golgi regions have also been shown to display projecting tubules. The concept of a “trans-Golgi network” was introduced by Griffiths et al. to discuss a compartment where the three major classes of proteins that move through the Golgi might be sorted and packaged for export; proteins destined for the plasma membrane (via constitutive vesicles) would be separated from proteins to be secreted (via regulated granules) and those destined for lysosomes (through clathrin-coated vesicles). This compartment was described as a tubular reticulum on the trans side of a Golgi stack. Contrary to this description, we do not see a reticulum of interconnected trans tubules, neither in this model nor in our earlier study of the TGN , nor in other reconstructions of fast-frozen, freeze-substituted material (data not shown). As with the CGN, we suspect that preparative methods may contribute to the extent to which Golgi cisternae appear reticulated. The TGN has usually been visualized with a specific structural marker: enzyme cytochemistry to reveal a phosphatase, photoconversion of a fluorescent lipid to visualize the trans Golgi more generally, or immunolocalization with a Golgi-specific antibody. All such work has been based on aldehyde-fixed material, so the possibility of membrane rearrangements during specimen preparation cannot be excluded. Moreover, cytochemical staining often involves diaminobenzidine, which is sufficiently hydrophobic to partition into lipid bilayers, expanding them artificially . Taken together, these results suggest that, whereas cytochemical staining may provide highly relevant data in terms of enzyme localization, such procedures are less than ideal for defining cisternal architecture. Our model is also at variance with the classical description of TGN in its paucity of tubules that project from trans-Golgi cisternae. This difference may be a result of our small sample size and represent only the specific regions that have so far been reconstructed. Other regions of the Golgi ribbon, in this or in other cells, may well show more tubules. Further work will be required to assess this possibility. Cisternae C6 and C7 are structurally different, both from each other and from the other cisternae of our model . The diameter and density of their fenestrae are distinct , their volumes are larger that those of C3–C5 , and their intimate association with trans ER is unique . They and C5 project tubules into the regions trans from the Golgi stacks. The most significant difference between these cisternae is that C7 buds exclusively clathrin-coated vesicles, while C5 and C6 bud only vesicles with nonclathrin coats. C7 is also unique in covering much of the trans side of the NCR. C6 is the most structurally variegated of all the cisternae in this model. It includes two unusually shaped bridges across the NCR, and it gives rise to two extensive tubules: one that curves around C7 and the trans ER to end with three budding profiles , and another that extends past all five of the preceding cisternae to end with a budding profile near the ERGIC . For all these reasons, we consider C6 and C7 to be distinct trans cisternae. The trans-reaching tubule on C5 adds a translike property to this medial cisterna, thereby giving it a transitional character. The idea of distinguishable trans cisternae is not new. A cornerstone of the GERL hypothesis of Novikoff et al. was that cytochemistry identified different enzymes in the two trans-most cisternae . When the dominant localization technology changed to immunogold labeling of frozen thin sections, however, differences in the trans cisternae were no longer observed . This may be due in part to the resolution of the technique: Golgi cisternae are separated by ∼20 nm, and the labeling method used has a resolution of about that magnitude . Therefore, even if a molecule is localized to a single cisterna, its appearance might “spill over” to the next cisterna. Clathrin is involved exclusively in packaging products destined for the endosomal/lysosomal pathway . The finding that C7 both uniquely and exclusively produces clathrin-coated buds indicates that this cisterna packages molecules for the lysosomal pathway. The presence of long tubules and extensive nonclathrin buds on the penultimate trans cisterna (C6) suggests that it is the most probable site for packaging molecules that are destined to the plasma membrane. Ladinsky et al. showed that individual tubules that are continuous with different trans cisternae produced buds that were either clathrin coated or covered with a lace-like coat, but never a mixture of the two. These observations suggested that sorting of molecules must occur before the formation of such TGN tubules, but they raised the question of whether a single cisterna might produce vesicles with only one or two coat types. Our new data indicate that each cisterna produces vesicles of only one coat type. This allows us to extend our hypothesis about the mechanism for exit from the Golgi to suggest that molecules destined for the plasma membrane are sorted to a different cisterna than those targeted for the endosomal/lysosomal pathway; from each of these cisternae they are then packaged for transport. In this model, two or more trans cisternae are involved in exit from the Golgi, so together they correspond to the TGN. As discussed above for the CGN, there does not appear to be a TGN compartment that is distinct from the trans cisternae and their tubules. This hypothesis about the mechanism of Golgi exit would be strengthened if we were able to distinguish the lacelike coat from other coat types, which we cannot. Three factors may have contributed to this problem. First, rapid freezing and freeze substitution, which preserves the complexity of cytoplasm with little if any extraction, may have obscured the lacelike coat. Coat structures are best seen in samples where cytoplasm has been extracted . Second, tannic acid was present in the freeze-substitution medium to enhance the contrast of membranes and coats. The deposition of tannic acid may have masked the fine detail of the lacelike coat, making it indistinguishable from COP coats. Third, the BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-ceramide used as a stain in the previous study may have enhanced, or even created, the appearance of the lace-like coat. Each of these factors is now being examined. It is important to note that there are other possible explanations for why the trans-most cisterna of our model exhibits only clathrin-coated buds. Temporal differences in cisternal activities could result in a cisterna's cycling between phases of sorting and packaging of molecules destined for different pathways. We think this explanation is unlikely because multiple trans cisternae form tubules, suggesting that multiple trans cisternae can function like the TGN in export from the Golgi . A close apposition of trans ER with the two trans-most Golgi cisternae is a prominent feature of fast-frozen/ freeze-substituted NRK cells and is commonly seen in thin sections of PtK and CHO cells preserved in a similar manner (data not shown). The apposition has also been described in numerous chemically fixed cells . Its roles in Golgi function should therefore be explored. We propose that this close apposition allows an exchange of lipids either to or from the trans-Golgi membranes by a nonvesicular, intermembrane, lipid-hopping mechanism. Precedent for such a hypothesis is found in the transfer of phosphatidylserine from ER to mitochondria for the production of phosphatidylethanolamine, a major component of mitochondrial membranes . Such a mechanism may also account for the movement of diacylglycerol from the trans Golgi to the ER, where it serves as a precursor for the generation of new phosphotidylcholine . A nonvesicular type of lipid recycling from the plasma membrane to the ER has been postulated to occur in plants, based both on fluorescent lipid redistribution studies and on the visualization of transient appositions between these two membrane systems in cryofixed cells . These factors, together with the obvious need for lipids to package exported proteins, encourage us to consider mechanisms for lipid transfer directly between the ER and the trans Golgi. Evidence for a lipid-hopping mechanism is found in the transfer rates of newly synthesized phospholipids from ER to the plasma membrane : t 1/2 = 2 min for phospholipids at 25°C, while newly synthesized cholesterol takes ∼10 min. The transit of phospholipids is thus too fast to occur via conventional membrane traffic from ER to Golgi to the plasma membrane, but it could be accomplished by the insertion of lipids directly at the TGN. Such transfer could be mediated by polypeptides like the antibacterial polymyxin B, which at very low mole fractions can form stable contacts between vesicles containing anionic phospholipids and stimulate lipid exchanges through these contacts at ∼300 molecules/s . We conclude that lipid transfer is a plausible reason for the close apposition between ER and trans-Golgi cisternae. The novel data presented here place structural constraints on the models now being considered for transit to, through, and from the Golgi complex. The hypothesis of tubular transport suggests that anterograde transport between successive cisternae is mediated by transient tubular connections . Since we found no connections between nonequivalent cisternae, nor any bridging tubules with branches to two or more cisternae, our data do not support this theory. Transport based on tubules with associated buds almost certainly occurs in the Golgi, but it seems to function in transport out of the Golgi in both the cis and trans directions. The fact that both cis- and trans-projecting tubules can pass one or more cisternae suggests that Golgi traffic need not visit all compartments on its way through or out of the Golgi. We surmise that more extensive tubules might also project from the trans-most Golgi cisterna at some time other than that of our freezing. All these tubules are likely to correspond to the ones seen in vivo, which have contributed to the concepts of CGN and TGN. It is more difficult to provide insight into the processes of vesicular transport, as vesicles are abundant in our model as well as in many published images. All cisternae have buds, indicating the formation of vesicles, so our data are consistent with vesicular traffic for both directions of intra-Golgi transport. There is also a wealth of vesicles at both the cis and trans faces of Golgi stacks, suggesting that both vesicles and tubules are involved in exit from the Golgi. The cisternal progression/maturation model posits that new cis/CGN cisternae are constantly being formed from VTCs (ERGIC), so indications of new cisternae should be seen on a frequent basis . The polymorphic ERGIC elements in this reconstruction are poorly organized and show no sign of fusion with the C1 cisterna or with each other to form a new cisterna. These elements may, however, represent VTCs just after their arrival at the cis-most cisterna. Delivery of additional VTCs, followed by their fusion and flattening beyond that seen in the ERGIC elements of our model, could eventually form a new cisterna. The remarkable patterning of ERGIC over the cis-most cisterna suggests this possibility and encourages further consideration of this model. Final characterization of the pathways for Golgi traffic will require work that combines reliability of specimen preparation and 3-D visualization, which characterize the current study, together with the identification of the specific molecular components that play key transport roles. Thus, our study forms a framework for future structural and experimental work that will focus on the locations and rearrangements of particular Golgi molecules. | Study | biomedical | en | 0.999996 |
10087260 | S. cerevisiae strains used in this study are listed in Table I . Yeast complete (YPD) and minimal media (SD) have been described previously . YNO medium contained 0.1% oleic acid, 0.05% Tween 40, 0.1% yeast extract, and 0.67% yeast nitrogen base without amino acids, adjusted to pH 6.0. When necessary, auxotrophic requirements were added according to Ausubel et al. . For induction of the CUP1 promoter CuSO 4 was added according to Marzioch et al. . For construction of pRS- PEX7 HA 3 , the SacI/KpnI fragment of YIp- PEB- HA 3 was subcloned into low copy plasmid pRS316 . YEp351- PEX14 was constructed by subcloning the SacI/BamHI fragment of pRS- PEX14 into YEp351 . YEp351- PEX13 was constructed by subcloning the XhoI/PstI fragment of pRS- PEX13 into YEp351 . For construction of Pex14pAXXA and Pex13pE320K, mutations were introduced by PCR using gene splicing by overlap extension . Primers used to construct Pex14pAXXA were: KU107, 5′-(GGAATTCGAGGCCTTATGAGTGACGTGGTCAGT)-3′, (nucleotides [nt] 1–18); RE9, 5′-(ATCCCTGTGGG C CAGCGTTG C TGGCATCGC)-3′, (nt 249–278; underlined nt represent introduced mutations); RE8, 5′-(GCGATGCCAGCAACGCTGGCCCACAGGGAT)-3′, (nt 249–278); and KU109, 5′-(GGGGATCCCGGGATACCTATGGGATGGAGTCTTC)-3′, . pRS- PEX14 served as template. Primers used to construct Pex13p-E320K were T3, 5′-(AATTAACCCTCACTAAAGGG)-3′; RE51, 5′-(CTCTGGGTTTTTTGGAACAAAATC)-3′, (nt 946–969); RE50, 5′-(GATTTTGTTCCAAAAAACCCAGAG)-3′, (nt 946–969); and T7, 5′-(GTAATACGACTCACTATAGGGC). pRS- PEX13 served as template. The PEX14 -PCR product was digested with EcoRI/ BamHI and subcloned into pBluescript SK(+) (Stratagene) resulting in plasmid pWG14/4. The PEX13 -PCR product was digested with XhoI/PstI and subcloned into pBluescript SK(+) resulting in plasmid pWG13/13. For complementation studies, pRS- PEX14 was digested with HpaI/HindIII and the internal fragment of PEX14 (nt 123–702) was exchanged by a HpaI/HindIII fragment of PEX14 AXXA from plasmid pWG14/4. The resulting plasmid pWG14/9 contained the PEX14 AXXA-ORF as well as 601 bp of the 5′- and 628 bp of the 3′-noncoding region of PEX14 . For complementation studies of pex13Δ , the 1.9-kb XhoI/PstI fragment of pWG13/13 containing the PEX13 E320K-ORF, as well as 356 bp 5′- and 381 bp 3′-noncoding region of PEX13 were subcloned in the yeast CEN-plasmid pRS351 resulting in pWG13/15. The two-hybrid assay was based on the method of Fields and Song . The ORFs of selected PEX genes were fused to the DNA-binding domain or transcription-activating domain of GAL4 in the vectors pPC86 and pPC97 . To construct the Gal4p-(BD)- Pex13p fusion, the EcoRI/SpeI fragment of plasmid pSP43D containing the complete ORF of PEX13 was subcloned into pPC86 resulting in pPC86- PEX13 . To construct the Gal4p(DB)- Pex14pAXXA fusion, the 1.1-kb EcoRI/SacII fragment from pWG14/4, containing the complete PEX14 AXXA-ORF was subcloned into EcoRI/ SacII-digested pPC86. The resulting plasmid was designated pWG14/6. The SalI/SacII of pWG14/6 contained PEX14 AXXA and was subcloned into SalI/SacII digested pPC97, resulting in plasmid pWG14/8. The PEX13 E320K ORF was amplified from plasmid pWG13/13 by PCR, using oligonucleotides 43fus1 5′-(GTGAATTCGGATCCATATGTCATCCACAGCAGTA)-3′ and the T7-primer (see above). The resulting PCR fragment was subcloned into a EcoRI/SpeI digested pPC86, resulting in plasmid pWG13/18. Amino acids 286–386 of Pex13pE320K were amplified by PCR using primers 37hyb1 5′-(TCCAGAATTCGGATCCTACAGACCTCTGGAACCATA)-3′, T7, and pWG13/13 as templates. The PCR product was subcloned with EcoRI/SpeI into pPC86, resulting in plasmid pWG13/16. Subsequently, pWG13/16 was digested with SmaI/SpeI, and the excised PEX13 E320K fragment was subcloned into SmaI/SpeI digested pPC97, resulting in plasmid pWG13/19. Constructions of the Gal4p-AD-Pex5p, Gal4p-DB-Pex7p, and Gal4p-AD-Pex14p fusion proteins have been described previously . Cotransformation of two-hybrid vectors into the PCY2 and HF7c was performed according to Gietz and Sugino . Double transformants were selected on SD synthetic medium without tryptophane and leucine. β-galactosidase activity of transformed cells was determined by a filter assay described by Rehling et al. , using X-Gal ( GIBCO BRL ) as substrate. His auxotrophy of transformed HF7c was determined by growth on selective plates lacking leucine, tryptophane, and histidine, but containing 10 mM 3-aminotriazole. To delete PEX5 , PEX13 , PEX14, and PEX17 in the yeast two-hybrid reporter strains PCY2 and HF7c , as well as the deletion of PEX7 and PEX13 in wild-type UTL7A, PEX14 in pex17Δ , PEX17 in pex13Δ , and PEX5 in pex14Δ , the kanMX4 gene was used as a selective marker for insertion into the genomic locus . Deletion cassettes containing the kanMX4 gene and the 5′ and 3′ untranslated regions of the corresponding ORFs were constructed by PCR using pFA6a- kanMX4 as a template. To generate the deletion cassettes for the PEX5 gene deletion, the primer sets KU301, 5′-(TATACATCAATAAACAATATATCATAACACATGGACGTACGTACGCTGCAG- GTGGAC)-3′ and KU302, 5′-(TGATGCGAGAACATAAAATTGCGGAGAACCATATCAATCGATGAATTCGAGCTCG)-3′ were used. For the PEX13 gene deletion, the primer sets KU274, 5′-(TATCTATAAATATCAAGGGGATTCTATACTATAACAATACCTGCGC-GTACGCTGCAGGTCGAC)-3′ and KU275, 5′-(TTTACTATATATATATGCGAATATATG TG TGCAAATATTGATGCAATCGATGA- ATTCGAGCTCG)-3′ were used. For the PEX7 gene deletion, KU371, 5′- (GTTAACGGAC TAT CATC TAAC TTTTTGCATAATTTATACAACATGCGTACGCTGCAGGTCGAC)-3′ and KU372, 5′-(GTTTAAATAATGCAAAAAATTTGTGTAAAAAGAATATGTGTCAACATC- GATGAATTCGAGCTCG)-3′ were used. For PEX14 gene deletion, the primer sets KU289, 5′-(GAAAACTCAAGTAAAACAGAGAAGTTGTAAGGTGAATAAGGACGTACGCTGCAGGTCGAC)-3′ and KU290, 5′- (AATTACAATTTCCG TTAAAAAAC TAATTACTTACATAGAAATTGCGATCGATGAATTCGAGCTCG)-3′ were used. Finally, for a PEX17 gene deletion the primer sets KU251, 5′-(TCCATCATTCT GATAAGCAGAACCACG TAAGGCAGAC TAAAATCCG TAC-GCTGCAGGTCGAC)-3′ and KU273, 5′-(ACGTGCACTAGAGCGTTTTAAATTCAATGCTATTATTTTTGATTGATCGATGAATTCG- AGCTCG)-3′ were used. For construction of the double knockout strain pex5/14Δ , the PEX14 locus was replaced by the loxP-kanMX4-loxP cassette . The PEX14 deletion cassette was constructed by PCR with the primer set KU289, 5′-(GAAAACTCAAGTAAAACAGAGAAGTTGTAAGGTGAATAAGGACGTACGCTGCAGGTCGAC)-3′ and RE24, 5′- (CAATTTCC GTTAAAAAAC TAATTAC TTACATAGAA TTGCGATAGGCCACTAGTGGATCTG)-3′ with plasmid pUG6 as template . The loxP-kanMX4-loxP cassette was integrated into the PEX14 locus and excised afterwards by the CRE recombinase expressed from plasmid pSH47 . Subsequently, PEX5 was deleted as described above. PCRs and selection for geneticin resistance of transformants were performed according to Wach et al. . Authenticity of each gene deletion by integration of the kanMX4 gene was confirmed by PCR. Spheroplasting of yeast cells, homogenization, and differential centrifugation at 25,000 g of homogenates were performed as described previously . For subfractionation by flotation gradient centrifugation, solid sucrose was added to a cell free extract, for a final concentration of 50% (wt/wt). 7 ml of this extract was overlaid with 5 ml 46% (wt/wt), 15 ml 38% (wt/wt), and 3 ml 25% (wt/wt) sucrose in gradient buffer (5 mM MES, 1 mM EDTA, 1 mM KCl, 0.1% [vol/vol] ethanol). Gradients were centrifuged in a Sorvall-SV288 rotor at 20,000 rpm for 4 h. 27 fractions were collected from the bottom and processed for enzyme measurement and Western blotting. 3-oxoacyl-CoA thiolase (EC 2.3.1.16), catalase (EC 1.11.1.6), and fumarate hydratase (fumarase: EC 4.2.1.2) were assayed according to published procedures . The peroxisomal peak fractions of a sucrose density gradient were pooled and subsequently diluted fivefold in gradient buffer . Peroxisomes were concentrated by centrifugation at 25,000 g for 30 min. The resultant pellet was resuspended in homogenization buffer supplemented with 50 mM KCl, but without protease inhibitors. Equal amounts of isolated peroxisomes were incubated for 10 min on ice with increasing amounts of proteinase K. The proteinase was inhibited by the addition of 4 mM PMSF. The proteins were then precipitated with TCA and the samples were processed for SDS-PAGE. The protein fusion and purification system of New England Biolabs was used for overexpression of a maltose-binding protein–SH3 domain fusion protein (MBP-SH3). Amino acids 286–386 of Pex13p comprising the SH3 domain of Pex13p were fused to the COOH terminus of the Escherichia coli MBP . MBP-SH3 was expressed from pMAL-SH3 in E . coli strain TG1. The expression was induced with 0.3 mM isopropyl-β- d -thiogalactopyranoside (IPTG), and affinity purification of the MBP-SH3 fusion protein on amylose resin was performed according to the manufacturer's protocol. Polyclonal antibodies were raised against the fusion protein (Eurogentec). Rabbit polyclonal anti-Pex11p antibodies were raised against a synthetic peptide (KAKSQSQGDEHEDHK) corresponding to amino acids 162–176 generated by Eurogentec. Antithiolase , anti-Pcs60p , anti-Pex3p , anti-Pex5p , anti-Pex14p , anti-Pex17p , and anti–fructose 1,6 bisphosphatase have been described previously. Anti–rabbit or anti–mouse IgG-coupled HRP (Nycomed Amersham ) was used as a secondary antibody, and blots were developed using the ECL-system (Nycomed Amersham ). Western blot analyses were performed according to standard protocols . Coimmunoprecipitation experiments were performed as described in Rehling et al. with the exception of omitting the 35,000 g sedimentation step. Immunofluorescence microscopy was performed according to the procedure of Rout and Kilmartin , with modifications previously described in Erdmann . Rabbit antisera against thiolase were used at dilutions of 1:3,000. For detection, 6 μg/ml solution of CY3-conjugated donkey anti–rabbit (Jackson ImmunoResearch Laboratories) was used. HA-tagged Pex11p was detected with monoclonal 12CA5 antiserum (BAbCO; dilution of 1:1,000). Yeast cells were grown on 0.3% SD medium to late log phase and subsequently for 15 h in YNOD (0.1% dextrose, 0.1% oleic acid, 0.05% Tween 40, 0.1% yeast extract, and 0.67% yeast nitrogen base). Cells were washed with dH 2 O and 1 g was used per sedimentation. 3 ml of buffer A (50 mM Tris-HCl, pH 7.5, 50 mM NaCl), protease inhibitors (0.02% PMSF [Serva], 15 μg/ml bestatin, 1.5 μg/ml pepstatin, 1 μg/ml leupeptin, 0.1 μg/ ml chymostatin [ Boehringer Mannheim ], 0.21 mg/ml NaF), and 3 g glass beads (0.5 mm) were added to the cells. Breakage was achieved by vortexing for 4 min . Samples were filtered through cotton wool, and the filtrate was transferred to Corex tubes and centrifuged at 1,000 g for 30 min. Supernatants were normalized for protein and volume, and membranes were sedimented at 200,000 g for 30 min (Sorvall AH650, 40,850 rpm) through a cushion of 0.25 M sucrose in buffer A. The resulting pellet was resuspended in buffer A plus protease inhibitors corresponding to the volume of the supernatant. Aliquots of the samples were analyzed by SDS-PAGE. It has been reported that the import receptors Pex5p and Pex7p interact with each other in the yeast two-hybrid system, which opened the possibility that both proteins may form a heteromeric cytosolic signal recognition complex . However, the yeast two-hybrid system does not necessarily distinguish between a direct and indirect binding of two S . cerevisiae proteins, as endogenous proteins may contribute to the observed interaction. As Pex14p can bind both import receptors, we investigated whether the Pex5p/Pex7p interaction is still observed in a yeast two-hybrid reporter strain deleted for the genomic copy of PEX14 . As shown in Fig. 1 , the interaction of the two import receptors strictly depended on the presence of Pex14p which may be required for the activation of either receptor, or it may function as a bridging molecule between Pex5p and Pex7p. Deletion of genes encoding the three components of the docking machinery known to date, Pex13p, Pex14p, or Pex17p, results in an import defect for both PTS1 and PTS2 proteins. One possible explanation could be a functional overlap of the proteins in PTS1- and PTS2-dependent protein import into peroxisomes. This prediction prompted us to search for additional Pex7p-binding proteins at the peroxisomal membrane. We expressed a functional mycPex7p fusion protein in wild-type, pex17Δ , pex14Δ , and pex13Δ cells, and determined the localization of the protein by subcellular fractionation studies. Fractions were analyzed for the presence of mycPex7p and for peroxisomal membrane markers Pex14p and Pex13p, as well as for cytosolic Fbp1p as a control for proper separation. Pex14p and Pex13p, but not Fbp1p, pelleted, indicating the complete sedimentation of cytosol-free peroxisomal membranes . As reported previously , mycPex7p was predominantly found in the soluble fraction in wild-type cells, while a low but significant amount was detected in the membrane fraction. A decrease of mycPex7p in the pellet fraction of pex14Δ cells suggests that the majority of sedimentable Pex7p associates with membranes in a Pex14p-dependent manner. However, in pex14Δ cells a significant amount of mycPex7p was detected in the membrane pellet fraction , indicating that next to Pex14p additional binding factors for Pex7p exist at the peroxisomal membrane. To determine the binding partners of Pex7p, we isolated mycPex7p from wild-type and various pex mutant strains by immunoprecipitation, and immunoblotted for the presence of selected peroxins. As reported previously, we found Pex5p, Pex13p, Pex14p, and Pex17p associated with mycPex7p when the receptor was precipitated from wild-type or complemented pex7Δ cells . Comparison of the constituents of the precipitates revealed five interesting observations. First, in pex14Δ and pex5Δ / pex14Δ strains, Pex13p still coimmunoprecipitated with mycPex7p , suggesting that Pex13p associates directly or indirectly with Pex7p. Moreover, this result indicated that neither Pex14p nor Pex5p is required for the formation of this subcomplex of Pex13p and Pex7p. Second, the amount of Pex5p in the precipitate from pex14Δ cells was drastically reduced, while the amount in Pex13p remained essentially unchanged . This result supports the notion that the amount of Pex5p bound to Pex13p does not determine the stoichiometry of the Pex13p-Pex7p subcomplex. However, it also suggests that Pex13p may not bind both import receptors equally at the same time. Third, Pex13p, Pex14p, and Pex5p still coimmunoprecipitated with Pex7p in pex17Δ cells . Obviously, Pex7p is associated with components of the peroxisomal translocation machinery in the absence of Pex17p, suggesting that the presence of Pex17p is not a prerequisite for docking of Pex7p to the peroxisomal membrane. Fourth, the lack of Pex17p in the coimmunoprecipitate from pex14Δ cells , suggests that Pex14p is required for the association of Pex17p with the complex, and is consistent with the assumption that Pex17p binding to the complex may be via Pex14p. However, this observation must be interpreted with care since the pex14Δ cells contain much less immunologically detectable Pex17p . Finally, the amount of Fox3p that coimmunoprecipitates with Pex7p drastically increases in mutants with an import defect for PTS2 proteins ( pex17Δ , pex13Δ , pex14Δ , and pex5Δ / pex14Δ ) relative to the strains unaffected in this pathway (wild-type and pex5Δ ). Since the total amount of both proteins is similar in all strains , it seems unlikely that the observed Pex7p/Fox3p complex has formed in vitro after cell disruption. A simple explanation for this may be that the high cytosolic concentration of thiolase in the import mutants results in greater occupation of the PTS2 receptor. To exclude the possibility of nonspecific coprecipitation of proteins, we checked the precipitates for the presence of peroxisomal membrane proteins Pex11p and Pmp35p (putative peroxisomal ATP-transporter; Erdmann, R., unpublished observations). These proteins were not detected in any of the samples, indicating the specificity of the observed interactions (data not shown). The observed in vivo association of Pex7p with Pex13p in cells lacking Pex14p and Pex5p encouraged us to analyze the interaction of these proteins in more detail. In previous experiments, only fragments of Pex13p were used to address whether Pex13p binds to Pex7p, and so far they have not indicated an interaction between these two proteins . To revisit this possibility, we analyzed the interaction of the full length Pex13p with Pex7p in the yeast two-hybrid system. The results shown in Fig. 4 A reveal that the full length Pex13p is indeed able to interact with the PTS2-receptor Pex7p. The controls included show that coexpression of either of the fusion proteins alone did not support transcription activation of the reporter genes. To analyze whether the observed Pex13p/Pex7p two-hybrid interaction depends on known binding partners for Pex13p, tests were also performed in isogenic pex5Δ and pex14Δ strains . Furthermore, we analyzed the association of Pex7p with a mutated Pex13pE320K in a pex5Δ mutant . Because Pex13pE320K lost the ability to interact with Pex14p in the yeast two-hybrid system , this experiment was expected to monitor the Pex13p/Pex7p interaction upon simultaneous elimination of the Pex14p and Pex5p influence. As shown in Fig. 4 , these two-hybrid analyses did not reveal an influence of Pex5p or Pex14p on the Pex13p/ Pex7p interaction. No difference was observed independent of whether the Pex7p/Pex13p interaction was analyzed in wild-type, pex5Δ , or pex14Δ strains , or for the Pex7p/Pex13pE320K interaction in pex5Δ cells . These results indicate that neither Pex14p nor Pex5p is required for the in vivo interaction of Pex7p with Pex13p, and therefore are in agreement with results obtained in the coimmunoprecipitation experiment . The two-hybrid interaction of the complete Pex13p with Pex14p is only detected by histidine prototrophy , indicating that regions NH 2 -terminal of the SH3 domain of Pex13p may weaken the interaction of these proteins in the two-hybrid system. Mutant cells lacking Pex7p are characterized by their inability to grow on oleic acid as the sole carbon source and by mislocalization of peroxisomal thiolase to the cytosol . Expression of a COOH-terminally HA-tagged Pex7p from the low copy plasmid pRS PEX7 -HA 3 leads only to a partial complementation of the pex7Δ mutant phenotype . This is indicated by the inability of the transformants to grow on oleic acid plates and a reduced ability to import Fox3p (thiolase) into peroxisomes. The latter is evident by the pronounced cytosolic mislocalization of this protein . This mutant phenotype of pex7Δ [pRS PEX7- HA 3 ] was employed to investigate whether overexpression of Pex7p-binding partners may suppress a defect in Pex7p function. Cells expressing Pex7p-HA 3 were transformed with multicopy plasmids containing either PEX14 or PEX13 under the control of their own promoters. As judged by their growth characteristics on oleic acid medium and by the fluorescence pattern for thiolase , overexpression of PEX13 , but not PEX14 , rescued the mutant phenotype caused by the defective Pex7p-HA. Even though the suppression was not as efficient as complementation with the wild-type PEX7 , this observation demonstrates that Pex13p can suppress the mutant phenotype of pex7Δ [pRS PEX7- HA 3 ], providing genetic evidence for an interaction between Pex7p and Pex13p. Pex13p is an integral peroxisomal membrane protein and the cytosolic orientation of the COOH-terminal SH3 domain was shown previously in human fibroblasts and Pichia pastoris . However, the COOH-terminal SH3 domain alone is not sufficient to interact with Pex7p, suggesting that regions NH 2 -terminal to the SH3 domain are involved in this association. To address whether the NH 2 terminus of Pex13p is localized to the lumen of peroxisomes or to the cytosol, we analyzed the accessibility of an NH 2 -terminally myc-tagged Pex13p to exogenously added protease K. The tag has been shown previously not to affect the function of Pex13p . Thus, the topology of the myc-tagged Pex13p is likely to reflect the in vivo situation for the wild-type protein. As judged by immunoblot analysis, both the NH 2 -terminal myc-tag as well as the SH3 domain of Pex13p were rapidly degraded by the protease . Intraperoxisomal thiolase remained stable under these conditions and was only degraded in the presence of detergents (data not shown). From this data, we conclude that both the NH 2 terminus and the COOH-terminal SH3 domain are exposed to the cytosol. This result also implicates the presence of an even number of transmembrane spans within Pex13p. Pex13p interacts with Pex14p via its COOH-terminal SH3 domain ; however, both proteins can interact with Pex7p independently. The latter is in agreement with the assumption that Pex13p and Pex14p contribute to distinct docking sites for Pex7p at the peroxisomal membrane. Since Pex14p is a peripheral membrane protein, two questions arise: How is it associated with the peroxisomal membrane, and Does Pex13p contribute to its localization? In an attempt to identify proteins required for the targeting and binding of Pex14p to the peroxisomal membrane, we analyzed the subcellular localization of Pex14p in different pex -mutant cells . The congruent fluorescence patterns for Pex14p and the peroxisomal membrane marker Pex11p in pex17Δ cells indicate a peroxisomal localization of Pex14p. This observation suggests that Pex17p is not required for the targeting of Pex14p to the peroxisomal membrane. In contrast, no congruent fluorescence patterns were observed in pex13Δ cells. Since the HA-tagged Pex11p is known to be targeted to peroxisomal membrane ghosts in pex13Δ cells , the lack of congruence suggests that the majority of Pex14p is mislocalized. To confirm this result by independent means, we performed a flotation of wild-type, pex13Δ , and pex17Δ homogenates in sucrose gradients . Gradients were designed such that peroxisomal membrane ghosts would float to the upper fractions of the gradient, whereas intact peroxisomes would predominantly remain in the loading zone. In pex13Δ cells, the peroxisomal membrane markers Pex3p and Pex11p were predominantly found in fractions that correspond to the peroxisomal membrane ghosts. However, Pex14p was not detected in these fractions, but was found to cosegregate with mitochondrial fumarase. These data suggest that the peroxisomal membrane ghosts in pex13Δ cells lack Pex14p. Thus, the presence of Pex13p is a prerequisite for peroxisomal membrane association of Pex14p. Pex13p could be involved in targeting, or it could be required for binding or retention of Pex14p at the peroxisome. To clarify whether binding of Pex14p to the SH3 domain is a prerequisite for the peroxisomal targeting of Pex14p, we analyzed the subcellular localization of Pex14p under conditions in which this interaction is blocked. A proline-rich sequence which corresponds to a type II SH3 domain binding motif is present within the primary sequence of Pex14p . We substituted Pro87 and Pro90 of this putative binding motif (P P TL P HRDW) by two alanines (P A TL A HRDW). Remarkably, the mutated Pex14pAXXA still complemented the peroxisome biogenesis defect of pex14Δ cells (data not shown). We also introduced an E320K mutation in the reverse transcriptase loop (RT loop) of the SH3 domain of Pex13p. This mutation has been reported to result in the inactivation of Pex13p function . As shown in Fig. 8 , the mutated Pex14pAXXA had lost the ability to bind Pex13p in the yeast two-hybrid system while binding to Pex5p, Pex7p, and oligomerization of the protein was unchanged. Also the E320K mutation of Pex13p abolished the two-hybrid interaction of the SH3 domain of Pex13p with Pex14p . These results suggest that strong interactions between Pex14p and the SH3 domain of Pex13p are dependent on the PXXP motif within Pex14p, as well as on the RT loop of the SH3 domain of Pex13p. Next, we analyzed the Pex14pAXXA association with peroxisomal membrane ghosts of pex14Δ/pex17Δ double mutants which were predicted to contain peroxisomal membrane ghosts even upon complementation of the pex14Δ mutation. In addition, we analyzed whether peroxisomal membrane ghosts that harbor mutated Pex13pE320K still contain Pex14p. The subcellular localization of Pex14p, Pex14pAXXA, and peroxisomal membrane markers was analyzed by double immunofluorescence microscopy and flotation analysis. Colocalization was observed for HA-Pex11p and Pex14pAXXA in pex14Δ cells, as well as for HA-Pex11p and Pex14p in pex13Δ cells expressing Pex13pE320K, indicative of peroxisomal membrane association of these proteins . These results were corroborated by flotation analysis which revealed that Pex14pAXXA was associated with the fraction containing the peroxisomal membrane ghosts of pex14Δ / pex17Δ , as were Pex14p in pex13Δ / pex17Δ cells expressing Pex13pE320K . These observations suggest that Pex14p is associated with peroxisomes and peroxisomal membrane ghosts independent of interaction between the proline-rich motif of Pex14p and the RT loop in the SH3 domain of Pex13p. Interestingly, the fractionation of pex13Δ / pex17Δ [ PEX13 E320K] shows that, although the RT loop of the SH3 domain of Pex13p is not absolutely required for the targeting of Pex14p to the membrane of peroxisomal ghosts, it appears to enhance or stabilize the targeting, as only Pex14p trails through the gradients of this mutant strain . The peroxisomal membrane protein Pex14p has been reported to bind both the PTS1 and the PTS2 receptor, which led Albertini et al. to the conclusion that Pex14p may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways at the peroxisomal membrane. Here, we report that Pex13p is also involved in both the PTS1- and PTS2-dependent protein import into peroxisomes. Pex13p interacts directly or indirectly with the PTS2 receptor Pex7p and overexpression of Pex13p suppresses the protein import defect caused by a functionally impaired epitope-tagged Pex7p. Pex13p is also shown to be required for the peroxisomal association of Pex14p; however, evidence is provided that the SH3 domain of Pex13p may not represent the only binding site for Pex14p at the peroxisomal membrane. The SH3 domain of Pex13p has been reported to interact with the PTS1 receptor Pex5p and with Pex14p . A mutation in the RT loop of the SH3 domain of Pex13p, as well as a mutation of a putative class II SH3 ligand motif of Pex14p abolished the two-hybrid interaction of both proteins , supporting the notion of a typical SH3 domain-ligand interaction between Pex13p and Pex14p. Interestingly, although the E320K mutation of the RT loop of the SH3 domain of Pex13p abolishes its two-hybrid interaction with Pex14p, the mutated SH3 domain still interacts with Pex5p . Accordingly, we conclude that there are distinct binding sites for both Pex5p and Pex14p within this domain or adjacent regions contained within the construct used for the assay. Remarkably, neither the E320K mutation of the SH3 domain of Pex13p nor the mutation of the proline-rich motif of Pex14p prevented the peroxisomal localization of Pex14p . This observation suggests that the binding of Pex14p to the SH3 domain of Pex13p is not absolutely required for the targeting and binding of Pex14p to peroxisomes. Why then does the absence of Pex13p lead to the mistargeting of Pex14p ? One possibility is that Pex13p is a component of a protein complex at the peroxisomal membrane that may disintegrate in the absence of the entire protein, but remains stable without the SH3-dependent interaction between Pex13p and Pex14p. Association of Pex14p with the complex may not require a direct interaction with Pex13p, but may be mediated by other components of the complex. The simplest explanation for our observations on the Pex13p/Pex14p interaction is the existence of an as yet unrecognized binding partner for Pex13p that may also provide the binding site for Pex14p at the peroxisomal membrane. This missing link, however, is not Pex17p. It is true that Pex17p is another binding partner of Pex14p, but our data suggest that Pex17p is not required for association of the Pex13p/ Pex14p/Pex5p/Pex7p complex, as all these components can efficiently coprecipitate in the absence of Pex17p . Moreover, we found no Pex17p in a precipitate from pex14Δ cells that still contains Pex13p and Pex7p , leading to two conclusions. First, a subcomplex of Pex13p and Pex7p can form in the absence of Pex14p and Pex17p, and second, Pex14p is required for the association of Pex17p with the complex. The latter may be explained by the assumption that Pex17p is bound to the complex via Pex14p. The amount of Pex7p in the membrane sediment of pex14Δ cells is significantly lower than in wild-type or pex13Δ cells , suggesting that Pex14p may contribute to the majority of the total binding capacity of the peroxisomal membrane for the PTS2 receptor. However, a significant amount of Pex7p was sedimented in the absence of Pex14p . Interestingly, in cells lacking both Pex13p and Pex14p, no Pex7p was found in the membrane pellet, which suggests that Pex13p contributed to the remaining Pex7p associated with peroxisomal membranes of pex14Δ cells (data not shown). This result, however, has to be interpreted with care since the double deletion of PEX13 and PEX14 did result in a significant decrease in immunologically detectable Pex7p (Girzalsky, W., and R. Erdmann, unpublished observations). The observations that Pex13p and Pex7p interact in the two-hybrid system and can be efficiently coimmunoprecipitated indicate that the proteins interact in vivo . Whether Pex13p directly binds Pex7p remains to be shown. Attempts to demonstrate direct binding of the proteins by coimmunoprecipitation of in vitro translated proteins were unsuccessful (data not shown). Our data do not exclude the existence of a bridging protein which would directly interact with both Pex13p and Pex7p, a requirement fulfilled by Pex14p. However, two observations indicate that the hypothetical bridging protein is not one of the known binding partners for Pex13p. First, the Pex7p/Pex13p interaction is also observed in the absence of these proteins , and second, the COOH-terminal SH3 domain alone is sufficient for the Pex13p/ Pex14p and Pex13p/Pex5p two-hybrid interaction, but not for the interaction of Pex13p with Pex7p . A direct interaction of Pex13p and Pex7p is further suggested by the genetic suppression of the defect caused by a functionally compromised HA-tagged Pex7p by overexpression of Pex13p . As discussed above, a Pex5p/Pex7p two-hybrid interaction is not observed in pex14Δ . At first, this observation seems rather surprising, since both Pex5p and Pex7p independently interact with Pex13p in the two-hybrid system . One could imagine that Pex13p may serve as a bridging molecule between the import receptors to mediate an indirect binding which could have emerged in the two-hybrid system. However, the amount of Pex5p simultaneously associated with the Pex7p-Pex13p complex may be too low to give a positive response. In support of this assumption, the amount of Pex5p coimmunoprecipitating with Pex7p in the absence of Pex14p is extremely reduced, despite the presence of significant amounts of Pex13p . Perhaps Pex13p does not usually associate simultaneously with both of the import receptors, or association is transient. The domain of Pex13 that interacts with Pex7p, as well as the side of the peroxisomal membrane where the interaction occurs, remains unknown. Furthermore, the intracellular localization of Pex7p in yeast is still a matter of debate. One group has reported that the protein is exclusively localized in the peroxisomal lumen , whereas others found the protein to be predominantly localized in the cytosol with a small amount associated with the peroxisomal membrane . Because the SH3 domain alone does not mediate the interaction with Pex7p, we suggest that regions NH 2 -terminal of the SH3 domain may be required for the interaction or contribute to the correct conformation of the binding site. Previously, the COOH-terminal SH3 domain has been reported to face the cytosol , and we found that both the NH 2 terminus and the COOH terminus of Pex13p are exposed to the cytosol , suggesting that the protein traverses the membrane with an even number of membrane spans. In this respect, it is interesting to note that two regions which would fulfill the requirement for α-helical transmembrane segments are present in Pex13p . The interaction of Pex13p with Pex7p has far reaching implications for our understanding of protein import into the peroxisomal matrix. Why are there several binding sites for the import receptors at the peroxisomal membrane? One hypothesis suggests that the multiple interactions reflect the existence of an import cascade in which the cargo-loaded import receptors are transferred from one component of the import complex to the next . Our confirmation that at least two peroxisomal membrane proteins bind the receptors raises concerns about which functions as the docking protein for each of the import pathways. Experimental evidence that Pex13p may be the docking protein for the PTS1 receptor has been provided , but the unsolved questions stress the need for reliable in vitro systems to study the order of interactions during the process of peroxisomal protein import. | Study | biomedical | en | 0.999997 |
10087261 | The VSVG-tag (YTDIEMNRLGK) was inserted at the NcoI site located directly at the initiation codon at the 5′ end of the cDNA of rat MVP. The resulting cDNA was cloned into the EcoRI site in the mammalian expression vector pcDNA3 ( CLONTECH ). The engineered construct was named plasmid encoding VSVG-tagged MVP (pvMVP). PC12 cells (phaeochromocytoma cells of rat adrenal medulla origin) were grown in DME supplemented with horse serum (10%) and FCS (5%) at 37°C in the presence of 5% CO 2 . CHO cells (derived from ovary cells of the Chinese hamster, Cricetulus griseus ) were cultivated with Ham's F12 medium supplemented with FCS (10%) at 37°C in the presence of 5% CO 2 . PC12 cells were rinsed carefully with 10 ml electroporation buffer (123 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 6 mM glucose, 20 mM Hepes, pH 7.05). The cells were removed from the culture flask with 5 ml (two times) electroporation buffer and centrifuged at 300 g for 5 min. The supernatant was discarded and the cell pellet was resuspended in 800 μl electroporation buffer. For transfection, the cell suspension was mixed with 50 μg of plasmid DNA in a 4-mm electroporation cuvette. After incubation for 2–5 min at room temperature, electroporation was performed with the following parameters: 500 μF, 310 V, 129 Ω (BTX, Electro cell manipulator 600; Angewandte Gentechnologie Systeme GmbH). The transfected cells were resuspended thoroughly in 20 ml recovery medium (PC12 cell medium as described above supplemented with 3 mM EGTA) and incubated for 30 min at 37°C, 10% CO 2 . After centrifugation at 300 g for 5 min, the cells were resuspended in 14 ml of medium and grown in culture plates (diam 94 mm) for 48 h in the absence or presence of β-NGF (5 ng/ml; Sigma Chemical Co. ). For immunocytochemistry, cells were transferred to poly- d -lysine–coated Permanox coverslips (5 μg/cm 2 ; Sigma Chemical Co. ) 24 h after transfection. 1 d after seeding (5.0 × 10 4 cells/ml), the cells were processed for immunocytochemistry. Transfection of CHO cells was performed according to the protocol of PC12 cells. In contrast to PC12 cells, CHO cells were treated with a trypsin-EDTA solution (1×, GIBCO BRL ) for 1.5 min and resuspended in 30 ml CHO cell medium. After centrifugation at 300 g for 5 min, CHO cells were resuspended in 10 ml electroporation buffer and centrifuged a second time using the protocol for PC12 cells. The parameters for the transfection of CHO cells are as follows: 250 μF, 420 V, 129 Ω. For further stimulation of protein expression, sodium butyrate (6 mM) was added 16 h before assaying the cells. For immunoprecipitation of vMVP from CHO cells magnetic beads conjugated with anti–mouse IgGs (M-450; Dynal ) were used. The anti-VSVG IgG1–coated beads were prepared as follows: 10 8 beads were washed in 500 μl PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 7.4) containing 0.1% BSA. Beads were resuspended in 1 ml PBS/0.1% BSA and incubated with 2 μl of anti-VSVG antibody (clone P5D4; Sigma Chemical Co. ) for 30 min at room temperature. To remove excess antibody, beads were subsequently washed six times in 1 ml of PBS/0.1% BSA and stored at 4°C overnight. CHO cells (4 tissue culture dishes of 143 cm 2 ) transfected with the pvMVP plasmid were grown for 48 h. All subcellular fractionation steps were carried out at 4°C. After removal of media, cells were washed twice (5 ml/dish) in buffer A (150 mM NaCl, 1 mM EGTA, 0.1 mM MgCl 2 , adjusted to pH 7.4 with 10 mM Hepes/NaOH). Cells were mechanically detached using a cell scraper in buffer A (3 ml/dish) containing a cocktail of the following protease inhibitors: antipain, leupeptin, chymostatin (2 μg/ ml each), pepstatin (1 μg/ml), and benzamidine (1 mM). Culture dishes were rinsed again with 1 ml of buffer A containing the protease inhibitors. Cell suspensions were centrifuged for 5 min at 375 g yielding a pellet fraction (P1) and a supernatant fraction (S1); the latter was stored on ice. P1 was resuspended in buffer A containing the protease inhibitors and thoroughly homogenized by 12 (up and down) strokes in a 0.5-ml glass-Teflon™ homogenizer. The homogenate was centrifuged for 5 min at 1,000 g to yield a postnuclear supernatant (S2). A large amount of intracellular organelles of CHO cells was contained in the supernatant fraction (S1) resulting from a plasma membrane disruption upon detachment of cells. Therefore, S1 was subjected to high speed centrifugation (60 min at 180,000 g ) to yield the microsomal fraction, P3. P3 was homogenized in 300 μl of buffer A containing the protease inhibitors and combined with S2. The pooled fractions were layered on top of a linear sucrose gradient (4.8 ml, ranging from 0.2 to 1.6 M sucrose) and centrifuged for 60 min at 150,000 g in a swinging bucket rotor. Fractions of 300 μl were collected starting from the top of the gradient. The pooled fractions 7–9 (parent fraction) were used for immunoisolation. Isolation protocol was applied according to the manufacturer's instructions. In brief, anti-VSVG IgG1–coated beads were incubated for 1 h at 4°C with the vault-containing fractions. Beads were washed three times with 500 μl PBS/0.1% BSA. Elution of the immunocomplex was performed in 30 μl of sample buffer by boiling for 5 min in a water bath. Identically treated nontransfected CHO cells served as a control. The cellular localization of MVP was investigated using the affinity-purified polyclonal antibody against rat MVP . The anti-VSV–glycoprotein mAb was used to detect the tagged MVP. To compare the cellular distribution of rat MVP with synaptic vesicle markers and cytoskeletal elements the following antibodies and reagents were used: antibody against synaptophysin (1:50; pAb, G95; gift of Dr. Reinhard Jahn, Göttingen, Germany); antibody against VAMP-II/synaptobrevin-2 (1:100; mAb clone 69.1; Synaptic Systems); antibody against the synaptic vesicle protein, SV2 ; R820, pAb against glucose transporter 4 ; β-tubulin (1:40; clone 2-28-33); and phalloidin coupled with TRITC (1:40; both Sigma Chemical Co. ) to detect F-actin. Before fixation the living cells cultivated on Permanox chamber slides (Nunc) were washed twice with Ringer's solution (155 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 2 mM NaH 2 PO 4 , 10 mM glucose, 10 mM Hepes buffer, pH 7.2) at 37°C for 1 min each. The cells were fixed for 7 min in methanol at −20°C and rinsed three times (5 min each) with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 7.4). To prevent nonspecific binding of antibodies cells were incubated for 20 min with 5% BSA ( Sigma Chemical Co. ) in PBS. Primary antibodies were applied for 20 min in the presence of 1% BSA in PBS. After three rinses in PBS for 5 min each, secondary antibodies coupled with fluorescein (1:100; FITC) or rhodamine (1:50; TRITC; both Sigma Chemical Co. ) were applied under the same conditions. Finally, the cells were washed three times with PBS (5 min each). The cells were analyzed by using an Axiophot microscope ( Zeiss ) or a laser-assisted (TCS4D; Leica GmbH) true confocal scanner. SDS-PAGE was carried out on minigels (10% acrylamide). Immunoblotting was performed using the enhanced chemiluminescence system (ECL; Amersham ) according to the manufacturer's instructions. For analyzing length and yield of MVP transcripts, mRNA from CHO or PC12 cells was isolated using an mRNA kit (Oligotex Direct; Quiagen). To further investigate the influence of the differentiation state on the expression of the MVP transcript, PC12 cells were cultivated for 3 d in the presence of β-NGF (5 ng/ml; Sigma Chemical Co. ). In addition, mRNA from CHO cells transfected with pvMVP and stimulated with 6 mM butyrate was isolated. mRNA (800 ng each) was separated on a 1% agarose-formaldehyde gel, transferred onto Hybond filters ( Amersham Pharmacia Biotech ), and hybridized at 68°C in the presence of 5× SSC (0.75 M NaCl, 75 mM sodium citrate, pH 7.0) overnight. A 2.2-kb DIG-labeled RNA deduced from the 3′ end of a rat MVP cDNA clone, isolated from a rat brain Uni-ZAP XR library (Stratagene), was used as a probe. A 1.5-kb DIG-labeled RNA probe, deduced from a mouse β-actin cDNA clone (Stratagene), was used as a control. Vaults have been found in all eukaryotic cells analyzed so far with the exception of yeast. The MVP, the predominant member of vaults contributing to >70% of their mass, is abundant both in CHO and PC12 cells. By Western blot analysis using the polyclonal anti–rat vault antibody, one protein band of 104 kD is specifically detected in the total cell homogenates of both cell lines at a similar level . The antibody raised against rat vaults recognizes the MVP in all species investigated; from Dictyostelium to humans with molecular masses ranging from 92 to 110 kD indicating a high conservation of the protein. As judged by immunodetection the level of MVP in PC12 cells seems to remain constant over 14 d of culturing time. Moreover, the amount of MVP and its corresponding transcript in PC12 cells appear to be independent of NGF-induced differentiation as estimated by Western and Northern blot analyses (not shown). In contrast to the yields of immunodetected protein, the expression level of the MVP transcript, as revealed in Northern blots, seems to be much higher in PC12 than in CHO cells . To verify that equal amounts of intact mRNA from both cell lines were loaded, the hybridization signal of the β-actin transcript is shown for comparison (lower panel). In this context it is noteworthy to state that in PC12 cells the 2.2-kb RNA probe of a rat MVP cDNA clone (comprising 75% of open reading frame from the 3′ end) labels several additional bands albeit less intensely. This hybridization pattern was confirmed in several experiments and did not differ in NGF-treated and untreated PC12 cells (not shown). That the stronger recognition of the MVP-transcript in PC12 cells as compared to CHO cells is not only due to the usage of a rat mRNA probe is revealed in Northern blots using an electric ray mRNA probe yielding a strong hybridization signal with the PC12 MVP transcript (not shown). Thus, signal strength might indeed represent a higher expression level in PC12 than in CHO cells. In the human neuroblastoma cell line, SH-SY5Y, the amount of MVP transcript is lower as compared to PC12 cells and equals that of CHO cells (not shown). Furthermore, MVP transcript size is considerably larger in PC12 than in CHO cells (4.9 versus 3 kb). An MVP transcript size of 4.9 kb was also found in rat brain and human neuroblastoma cells (not shown). In T. marmorata the MVP transcript size was ∼2.8 kb . The cDNA clones isolated for sequencing rat, electric ray, or slime mold MVP were ∼2.8 kb in size. A correlation between differences in size and amount of MVP transcripts in various species is not conclusive. Immunocytochemical analysis demonstrates the abundance of MVP in both cell lines . MVP is almost uniformly distributed throughout the entire cytosol. In differentiated PC12 cells labeling of MVP can also be detected in the neuritic extensions including their tips . In double labeling experiments the resolution obtained by conventional indirect immunofluorescence technique proved to be limited. Therefore, we analyzed the cellular distribution of vaults by confocal laser scanning microscopy with 20 serial sections analyzed per cell. Double labeling of CHO cells using antibodies against MVP and β-tubulin demonstrates that vaults are often colocalized with microtubules . Vaults reveal a discrete and punctate distribution throughout the cytoplasm . The distribution of β-tubulin resembles the typical fiber system of microtubules . There is a considerable overlap in the distribution of the cytoskeletal fibers and vault particles. High magnification of the cell periphery of CHO cells indicates a predominant orientation of vaults along the cytoskeletal tracks of microtubules . Similarly, in differentiated PC12 cells treated with NGF for 72 h the distribution of MVP overlaps with that of β-tubulin . Especially in the neuritic extensions, the distribution of vaults and microtubules appears to be similar. Staining intensity for β-tubulin in neurites of PC12 cells appears very strong, indicating a dense packaging of microtubules. The evaluation of a putative colocalization of vaults and microtubules remains limited to the soma of PC12 cells where the localization of single particles and fibers can be differentiated. There the majority of vaults are located in the vicinity of microtubules . The distribution of MVP- and phalloidin-stained filamentous actin in CHO cells is clearly different. Whereas vaults reveal a punctate pattern in thousands of loci throughout the cytoplasm, the actin-based stress fibers are concentrated at the ruffling edges of lamellipodia and span the entire cell as long filaments . In the cell periphery, only a partial overlap of vault and actin filament localization can be observed. Similarly, in differentiated PC12 cells the distribution of vaults and actin filaments differs in the cell soma . However, there is an apparent identical staining pattern of MVP and actin in PC12 cells that is restricted to the most distal areas of neuritic extensions including the tips . To analyze the sorting and targeting of MVP in a narrow time window and to perform further double labeling experiments, we engineered a construct coding for MVP and an additional epitope recognized by an mAb. Transient transfection of CHO and PC12 cells was performed with the plasmid pvMVP encoding the viral tag VSVG attached to the NH 2 terminus of MVP (vMVP). To differentiate the recombinant protein from the endogenous one, two antibodies were employed. The mAb directed against the VSVG epitope only recognizes the recombinant protein, whereas the pAb against rat MVP detects both the endogenous and the recombinant MVP. Transfection efficacy of electroporation differed considerably between CHO and PC12 cells. About 30% of CHO cells expressed the recombinant VSVG-tagged protein, whereas only ∼5% of PC12 cells vMVP could be detected by immunocytochemistry. In CHO cells the expression of recombinant and endogenous MVP was evaluated by Western and Northern analyses. The amount of the vMVP transcript is much higher than that of the endogenous MVP transcript . Northern blot analysis reveals a 3.2-kb vMVP transcript. The shift in transcript size occurs because of the attached nucleotides encoding the viral epitope as well as additional nucleotides required for cloning strategies of the pvMVP construct . Treatment of transfected CHO cells with butyrate results in a further increase of mRNA expression . Consistent with the dramatic increase of mRNA expression, de novo synthesis of transiently expressed vMVP far exceeded the amount of endogenous MVP . Accordingly, butyrate treatment further stimulates synthesis of vMVP . To further analyze if recombinant MVP is sorted like endogenous MVP, we investigated the composition of vMVP-containing particles. We immunoisolated vMVP from pvMVP-transfected CHO cells using a anti-VSVG mAb coupled to anti–mouse IgG–coated magnetic beads as a solid support. A combined fraction containing the postnuclear supernatant and microsomes derived from transfected CHO cells (four confluent plates) was subjected to high speed glycerol gradient centrifugation. As analyzed by Western blotting the bulk of vMVP immunoreactivity is retained in fractions 7–9 corresponding to 12–15% glycerol . There is no significant shift of vMVP immunoreactivity compared to total MVP immunoreactivity. Total MVP immunoreactivity of pvMVP-transfected cells seems to shift slightly to denser fractions . Thus, all vMVP is particulate and no vMVP was found to be soluble (e.g., contained in light fractions in the upper part of the gradient). The sedimentation behavior of vMVP resembles that of endogenous MVP. However, some MVP immunoreactivity smears to denser fractions. This indicates an association of vaults with larger structures . The lack of immunoreactivity in this region of the gradient in the top frame may occur because of loading less material and a shorter exposure time. The partially purified enriched vault gradient fractions 7–9 were used as starting material for immunoisolation. The isolated immunocomplex contained vMVP as shown by Western blot using the same anti-VSVG antibody employed for immunoisolation . Immunoisolation of identically treated nontransfected CHO cells demonstrates specificity of the antibody reaction . Silver staining of the eluted immunocomplex reveals vMVP and the presence of additional proteins . The polypeptide pattern of the immunocomplex shows coeluted immunoglobulins (heavy chains ∼50 kD), trapped BSA (65 kD; added to prevent nonspecific binding), and several minor bands. This pattern was confirmed in several independent experiments. NGF-treated PC12 cells develop a neuronal phenotype with neurite outgrowth and the appearance of numerous synaptic vesicle-like vesicle. SV2 used as a marker for secretory organelles such as chromaffin granules as well as synaptic vesicle-like vesicles reveal an intense and punctate distribution in the perinuclear region and in the tips of neurites . Consistently, the synaptic vesicle marker, VAMP-II/synaptobrevin-2 present within the same organelles, displays an identical intracellular localization . Double labeling for the vesicle markers and MVP demonstrates a differential distribution of vault particles and secretory organelles in many areas of differentiated PC12 cells. However, in the perinuclear region and in the neurites, especially in the tips, a similar staining pattern of vaults and vesicles could be detected . CHO and PC12 cells analyzed by laser-assisted confocal microscopy and double labeling technique 48 h after transfection with the pvMVP plasmid demonstrate a high expression of recombinant vMVP . In the transfected cell lines vMVP is distributed like the endogenous MVP . Slight differences in staining intensity of the two antibodies are restricted to the perinuclear region where the recombinant protein appears to be highly concentrated, presumably due to massive de novo synthesis of vMVP and lower affinity of the polyclonal antibody . Staining of the recombinant protein yields the typical distribution of vaults with the punctate pattern throughout the cytosol. This indicates that vMVP is sorted into vault particles and targeted to the same cellular destinations as endogenous MVP . The recombinant protein is also transported to the cell periphery . Arrowheads depict neighboring cells that are either nontransfected and, thus, vMVP negative , or processes of adjacent cells exhibiting only low expression of vMVP . We compared the distribution of vaults with GLUT4 which is localized in a specific cellular subcompartment. It characterizes a distinct class of intracellular storage vesicles (named surface modifying vesicle) in fat and muscle cells as well as in a variety of transfected cell lines . A CHO cell line stably transfected with GLUT4 was transiently cotransfected with pvMVP. Fig. 5 D displays the high expression of vMVP as compared to the constitutively expressed GLUT4. Staining for GLUT4 shows the typical intracellular punctate pattern concentrated in the perinuclear area . The distribution of vMVP is different from that of GLUT4-containing vesicles. Although the distribution of vMVP is also high in the perinuclear region, the overlap with GLUT4 vesicles is only partial . Staining for both markers shows a punctate pattern, although the vMVP-containing loci seem to be larger. PC12 cells transfected with pvMVP and grown in the presence of NGF for 48 h demonstrate a high expression of vMVP uniformly distributed throughout the cytosol with the characteristic granular appearance . Double staining with anti-VSVG antibody recognizing only vMVP and anti-MVP antibody detecting recombinant and endogenous MVP reveals an almost identical distribution of immunolabeling . vMVP is targeted to the tips of neurites as endogenous MVP . A neighboring cell with low vMVP expression is depicted by an arrowhead. The synaptic vesicle marker P38/synaptophysin present exclusively on synaptic vesicle-like vesicle is located at the cell soma and transported to neurites in differentiated PC12 cells . The distribution of P38/synaptophysin-containing vesicles considerably overlaps with that of vMVP . In contrast to SV2 and synaptobrevin that are both localized on chromaffin granules as well as on synaptic vesicle-like vesicle, synaptophysin accumulates in the neurite swellings distant to the tips . An extension of an adjacent cell without vMVP expression is depicted by an arrowhead. About 100 copies of MVPs are contained in one single vault particle, whereas only a few copies of the minor constituents contribute to the inventory of vaults . MVPs share a unique domain structure containing an elongated α-helical COOH-terminal tail, numerous phosphorylation motifs, and three repetitive elements . Electron microscopy reveals that vaults isolated from rat liver, electric ray, rabbit, Xenopus , bullfrog, sea urchin, and the lower eukaryote D. discoideum are similar both in their dimensions and morphology . Upon subcellular fractionation MVP is entirely retained in a particulate fraction. As shown by negative staining of subcellular fractions, MVP is assembled into intact vaults . Transient transfection of the engineered construct encoding rat vMVP in CHO cells led to a high de novo protein synthesis of the recombinant protein. Although the transfection efficiency was ∼30%, the synthesis of the heterologously expressed protein by far exceeded the amount of the endogenous protein. To further evaluate the components of vault particles containing vMVP we performed immunoisolation of pvMVP-transfected CHO cells using the coexpressed viral tag as target epitope. Western analysis of subcellular fractions demonstrates that vMVP behaving like endogenous MVP is exclusively present in a large complex retained in a particulate fraction. The separation method used gave results in fractionation according to size. Thus, vMVP is likely to be assembled into structures with approximately the same size as vault particles. However, the quantity of the vMVP particles expressed precluded the analysis of their structure using negative staining electron microscopy. The immunocomplex mainly consisted of vMVP, immunoglobulins, and most likely, trapped BSA. In addition, we found several minor bands not characterized yet. Minor constituents of endogenous vault particles have been reported to be proteins of 54, 192, and 210 kD as well as a nontranslatable vRNA of 37 kD . Although bands of the isolated immunocomplex migrated at the expected sizes, additional bands of similar or even stronger intensities could not be assigned to the pattern of vault components reported so far . Thus, the composition of vMVP-containing vaults is not conclusive. The assembly of vault particles is probably mediated by the elongated coiled-coil domain at the COOH terminus of MVP . Preliminary experiments with truncated versions of MVP indicate that the COOH terminus is essential for vault assembly. The amount of overexpressed vMVP might far exceed the required cellular content of the minor vault components which gives rise to the formation of vault particles consisting of vMVP and lacking the other constituents. The contribution of the minor components in highly purified vaults from various species can vary . Consistently, vault formation is limited by the expression of MVP . Vaults consisting mainly of vMVP and lacking the others might be a valuable tool to study the relevance of the minor constituents for vault function. Immunocytochemical analysis of transfected CHO and PC12 cells reveals a dense punctate pattern of MVP and vMVP throughout the entire cytoplasm in both cell lines. The granular cytoplasmic appearance of MVP represents the distribution of vault particles. In rat fibroblasts, a punctate cytoplasmic pattern of vault distribution with thousands of vault loci has been reported . Both in CHO and PC12 cells, MVP and vMVP are targeted to the cell periphery. Expression of vMVP is detectable 6 h after transfection of cells. 48 h after transfection vMVP is localized in the neuritic tips of differentiated PC12 cells identical to endogenous MVP. In CHO cells stably transfected with GLUT4 and transiently transfected with vMVP, the localization of GLUT4-containing vesicles and vMVP differs. In contrast, the distribution of MVP/vMVP-containing vaults coincides with markers of secretory organelles in PC12 cells. Double labeling demonstrates a partial colocalization of vaults and secretory organelles in the soma of PC12 cells. Colocalization of vaults and vesicles is obvious in neuritic tips. Markers for secretory vesicles used in this study reside either exclusively on small electron-lucent synaptic vesicle-like vesicles (P38/synaptophysin) or also on large granules (SV2 and VAMP/synaptobrevin) shown recently by immunoelectron microscopical analysis . Using the same technique and antibodies against MVP or SV2, a high concentration of vaults and synaptic vesicles was also found in the synaptic terminals of Torpedo electromotoneurons near each other . Comparison of the distribution of vaults with microtubules in CHO cells reveals a similar pattern in corresponding immunofluorescent images. Vault puncta seem to be oriented along microtubules from the center to the cell periphery. Especially at high magnification, an overlapping distribution of microtubules and vaults in cellular extensions becomes obvious. These observations imply the possibility that vaults, as abundant cytoplasmic particles, are contained in microdomains structurally organized by microtubules and appear to be codistributed. A comparison of the distribution of vaults with filamentous actin in CHO cells reveals a divergent localization. The typical granular cytoplasmic vault distribution differs remarkably from that of polymerized actin organized in long stress fibers and concentrated at focal adhesion points of spreading CHO cells. In spreading fibroblasts, vaults are clustered in localized zones within lamellipodia at actin-rich ruffling edges . Furthermore, we compared the distribution of vaults with microtubules or filamentous actin in differentiated PC12 cells that resembled a neuronlike cell type. Overlay of vault and microtubule immunofluorescence in serial sections of PC12 cells yields an almost identical localization of both structures in neurites. In the soma of PC12 cells there is no apparent colocalization of vault particles and actin filaments. However, in the tips of neurites the distribution of vaults and actin fibers seem to overlap completely, yielding an identical pattern of immunostaining. Our immunocytochemical data suggest that there is a cellular transport of vaults mediated by microtubules in the cell soma and neuritic extensions and actin filaments in neuritic tips, where we found colocalization of vaults and secretory organelles. It has been reported that vaults copurify with microtubules in sea urchin preparations, but the two can be separated by further fractionation. Thus vaults are not considered to be microtubule-associated proteins . The codistribution of vaults with microtubules in cell soma and neurites and the occurrence of vault particles in neuritic tips indicate regulated active cytoplasmic transport. In the neuritic tips, the distribution of vaults resembles that of secretory organelles. In the electric ray, T. marmorata , vaults occur in the axon and are abundant in nerve terminals of electromotoneurons implying active axonal transport . The dynamic evidence is further supported by crush experiments of the electric nerves demonstrating time-dependent axonal transport with anterograde and retrograde accumulation of vaults . Cytoplasmic transport of membranous organelles has been studied in a variety of cells. The nerve axon is a good model system for studying organelle transport. Vesicular organelles are axonally transported using microtubules as cellular tracks and motor proteins . In addition to the microtubule-dependent mechanism, fast transport of organelles also occurs in the presence of actin filaments . Occurrence of polyribosomes and different types of RNA including mRNAs and nonmessenger RNAs in dendrites has been reported . There is evidence that mRNA is transported as part of a larger structure on cytoskeletal elements . Proteins encoded by dendritic mRNA belong to diverse classes representing kinases, cytoskeletal components, and receptors. The axonal compartment of mammals has been thought to be devoid of mRNA. Recently in the neurosecretory axons of the hypothalamo-neurohypophyseal system, mRNAs encoding vasopressin, oxytocin, prodynorphin, and a neurofilament protein species have been identified . mRNA anchoring to actin filaments (fibroblasts) and also to microtubules (neurons) has been demonstrated . Reports about transport of nonmessenger RNAs are rare. Neural BC1 RNA, a small polymerase III transcript of unknown function, is preferentially expressed in the brain and is distributed to neuronal dendrites . Moreover, BC1 RNA, a component of a 10S ribonucleoprotein particle, is transported in hypothalamo-neurohypophyseal axons . Interestingly, it has been reported that BC1 RNA contains elements capable of linking a subset of mRNAs to microtubules presumably by controlling RNA transport along dendritic microtubules . Little is known concerning the transport mechanisms of RNA particles. Kinesin antisense oligonucleotides inhibit the transport of myelin basic protein mRNA in processes of cultured oligodendrocytes . RNA was found to be associated with granules. In cultured neurons, RNA granules were transported in anterograde and retrograde direction. The motility had characteristics of an organelle that might be actively transported along cytoskeletal tracks . Vaults are by far the largest ribonucleoprotein particles transported to nerve endings and neuritic tips. Our data propose that in neuronlike cell type, vaults may have additional specialized function. Our findings provide new insights into the cellular distribution of an organelle, which recently has begun to attract attention in different areas of cell biology. The occurrence and abundance of this large cytoplasmic ribonucleoprotein particle in eukaryotic cells argue for an important general function. This is further supported by the high phylogenetic conservation of its main component, the MVP. Cytoplasmic transport of vaults may be required for the various physiological functions postulated so far. A clinical relevance of the human MVP in the prediction of multidrug resistance phenotype in numerous cancer cell lines is well documented. Upregulation of MVP and vaults is a predictor of the multidrug resistance phenotype, but the cellular mechanisms are not understood. Although there is no preferential localization of vaults to the nuclear envelope, a functional role in nucleocytoplasmic exchange has been proposed. An involvement of vaults in intracellular traffic might be mediated by binding to other cellular organelles or targets. Recently, an interaction of vaults with intracellular steroid hormone receptors has been reported . The active nucleocytoplasmic shuttle might depend on interaction with members of the nuclear receptor superfamily. To obtain insights in the regulation of intracellular transport of vaults on cytoskeletal tracks is a future challenge. The targeting of this large ribonucleoprotein particle to the cell periphery implies new aspects of the functioning of vaults. | Study | biomedical | en | 0.999997 |
10087262 | Strains used in this study are listed in Table I . Complete medium, YPD (1% yeast extract, 2% polypeptone, 2% dextrose) which contains 10 μg/ml Phloxine B (called YPDP), YES5 (0.5% yeast extract, 3% dextrose, and 75 μg/ml of adenine, histidine, leucine, and uracil), and modified synthetic EMM2 have been described . Standard procedures for S . pombe genetics were followed according to Moreno et al. . Gene disruptions are abbreviated by putting the symbol Δ before the gene name, e.g., Δ mok1 . Proteins are designated by an uppercase first letter, e.g., Mok1. As an initial screen collection of temperature-sensitive (ts) or cold-sensitive (cs) mutants (2,822 or 1,961, the restrictive temperatures were set at 36 and 20°C, respectively) were visually selected by calcofluor staining and morphological mutants with altered cell shape at the restrictive temperature were isolated. Four types of morphological mutants were observed, namely round or pear-like, elongated, bent or branched, and septation defective. Round or pear-shaped mutants were further screened for the supersensitivity to a protein kinase inhibitor staurosporine (STS, 1.5 μg/ml) for which Pck1 and Pck2 are two of the major targets . To exclude mutants that were nonspecifically supersensitive to a broad range of drugs, sensitivity to K-252a (3 μg/ml), which structurally related but has distinct biological targets to STS , was also examined. After mating of each strain, free spores were directly plated on four YPDP plates, three of which were incubated at 20, 29, or 36°C and the fourth was incubated on a YPDP plate containing 1.5 μg/ml STS at 29°C. If the difference in colony number between the plates growing under these conditions was >10 4 -fold, the two mutants were assumed to be allelic. Standard molecular biology techniques were followed as described . Enzymes were used according to the recommendation of the suppliers ( New England Biolabs Inc. ). Nucleotide sequence was determined by use of dideoxy-method . An S . pombe genomic library constructed in the vector pDB248 was used for the isolation of plasmids that complemented the ts mok1 mutant (DH664). 2 out of 20,000 colonies were capable of growing at 36°C and the segregation analysis showed that the Ts + phenotype was plasmid-dependent. Two different plasmids containing different, but overlapping, inserts were isolated. Subcloning analysis indicated that the internal SmaI site was essential for the complementing activity of pMK1100. When the 5.5-kb SmaI/BamHI fragment was subcloned from pMK1100, the resulting plasmid lost the complementing activity. However, Ts + colonies appeared from transformants containing pMK1101 at a frequency of ∼10 −3 . It was found that pMK1101 directed integration of the LEU2 marker into the genome via homologous recombination in these Ts + transformants. Subsequent analysis showed that no ts recombinants arose from 10 4 recombinants between this integrant and a wild-type strain. This result demonstrated that pMK1101 contains the mok1 + gene itself. Homology searching using Mok1 as query against the fission yeast genome database (Sanger Centre, Cambridge, UK) showed that there are four additional mok1 + homologues [designated mok11 + (SPAC23D3), mok12 + , mok13 + (c16D10S), and mok14 + (c63), respectively]. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/Genbank nucleotide sequence databases under the accession numbers: AB019183 ( mok1 + ), AB018380 ( mok11 + ), AB018381 ( mok12 + ), AB018382 ( mok13 + ), and AB018383 ( mok14 + ). Three different strategies were taken to disrupt the mok1 + gene by use of a PCR-generated fragment containing the ura4 + marker : one resulted in the complete deletion of the ORF (SKDP1), the second in deletion of a 1,764 bp NH 2 -terminal fragment (corresponding to amino acids 66–654) and the third in fusing the DNA encoding HA or GFP tags to the COOH terminus, which was accidentally found during the attempt to construct the tagged mok1 + gene. Correct disruption was verified by PCR. In both cases, tetrad analysis of the heterozygous diploid showed two viable (Ura − ) and two inviable spores, indicating that the mok1 + gene is essential for cell viability. The mok11 + , mok12 + , mok13 + , and mok14 + genes were also disrupted. Tetrad analysis of a heterozygous diploid yielded four viable spores, in which Ura + and Ura − segregates 2:2 in each case. This indicated that neither mok11 + , mok12 + , nor mok13 + gene is essential for cell viability. It was also found that double mok11mok12 and triple mok11mok12mok14 disruptions were viable. Three different fragments of the mok1 + gene were amplified with PCR and inserted in pREP1 or pREP2 ; a 7.2-kb fragment containing the whole ORF (5′-TTTGGATCCTATGCATGGTCTTCAAGGGTTATGTTTTAGA-3′ and 5′-TTTGGATCCCTAAGGACGACTAAGGTTTTCACGACGGAA-3′ were used for PCR, yielding pREP1-mok1), an NH 2 -terminal 3.4 kb containing amino acid residues 1–1,236 (5′-TTTAGGCCTCTACGAGACTACAGGAGTGACCTT-3′ and 5′-TTTGGATCCTATGCATGGTCTTCAAGGGTTATGTTTTAGA-3′ were used for PCR, yielding pREP2-mok1N) and a COOH-terminal 3.9 kb containing amino acid residues 1,017 to 2,410 (5′-TTTGGATCCTATGCATGGTCTTCAAGGGTTATGTTTTAGA-3′ and 5′-TTTGGATCCCTAAGGACGACTAAGGTTTTCACGACGGAA-3′ were used for PCR, yielding pREP1-mok1C). The nmt1 promoter was integrated in the genome in front of the initiator ATG of the mok1 + gene by a PCR-based gene targeting method . Fission yeast cells, in which the nmt1 promoter was integrated in front of the ORF of mok1 + (SKP103), were grown in liquid minimal medium at 29°C for 12 h and placed on a slide glass embedded in a slice (1 mm) of agar made of minimal medium supplemented with leucine. This slice was then overlaid with a coverslip and sealed with liquid sealer. The slide glass was set under the phase microscope ( Zeiss Co. ) at room temperature (22°C) and the cells were viewed with a chilled video-rated CCD camera connected to a computer . Photographs of live cells in a fixed field were taken every 30 min. Images were processed by use of Adobe ® Photoshop (version 4). Cultures of S . pombe cells were supplemented with U-[ 14 C]glucose (1 μCi/ml) and incubated for additional 4 h. To label cells overproducing mok1 + (SKP103 and SKP170), cultures were induced for 14 h in the absence of thiamine before addition of U-[ 14 C]glucose. Total glucose incorporation was monitored by measuring the radioactivity in trichloroacetic acid insoluble material. Mechanical breakage of cells was done as described and cell walls were pelleted at 1,000 g for 5 min. 100 μl aliquots of the total wall were incubated with 100 units of zymolyase 100T (Seikagaku Kogyo Co. Ltd.) or Quantazyme (Quantum Biotechnologies Inc.) for 36 h at 30°C. The samples were centrifuged and the supernatant and washed pellet were counted separately. The polysaccharides in the supernatant from the zymolyase 100T reaction were considered to be β-glucan plus galactomannan, and the pellet α-glucan, whereas that from the Quantazyme reactions was considered to be β-glucan and the pellet α-glucan plus galactomannan. Rabbit polyclonal anti-Mok1 antibody was prepared as follows. To express the fused Mok1 protein, the 0.9-kb fragment corresponding amino acid residues 1,583 to 1,904 (5′-TTTCATATGTCTCAACGTACCCGTGCTCGACTT-3′ and 5′-TTTGGATCCATCTCTGATTTCATTAAGTATTGT-3′ were used for PCR) was inserted into pET10c (Invitrogen Co.). Insoluble proteins were purified using Ni-NTA column. Two rabbits were injected with the Mok1 fusion protein (200 μg per injection). Immunoblotting was performed with crude sera or affinity-purified anti-Mok1 antibodies. Bacterially made Mok1 fusion protein was bound to nitrocellulose filters in order to purify anti-Mok1 antibodies from crude sera. Monoclonal anti–α-tubulin antibody ( Sigma Chemical Co. ) was also used. Horseradish peroxidase-conjugated goat anti–rabbit IgG, goat anti– mouse IgG (Bio-Rad Laboratories Ltd.) and a chemiluminescence system (ECL; Amersham Corp. ) were used to detect bound antibody. Fission yeast whole cell extracts were prepared using glass beads to disrupt yeast cells in TEG buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 30 mM NaCl, 1 mM DTT, 60 mM β-glycerophosphate, 15 mM p -nitrophenylphosphate, 0.1 mM NaF, 10 μg/ml soybean trypsin inhibitor, 20 μg/ml leupeptin, 50 μg/ml aprotinin, 2 μg/ml pepstatin, 1 mM PMSF, and 0.1 mM Na-orthovanadate). For the subcellular fractionation, cell extracts were microcentrifuged at 13,000 rpm for 15 min at 4°C in various conditions in order to separate soluble from insoluble fractions. Rhodamine-conjugated phalloidin (Molecular Probes Inc.) or monoclonal anti-chicken gizzard actin antibody (N-350; Amersham Corp. ) followed by Cy3-conjugated sheep anti–mouse IgG ( Sigma Chemical Co. ) was used to visualize actin as described previously . Rabbit polyclonal anti-Mok1 serum (1:10 dilution) or undiluted affinity-purified antibodies and Cy3-conjugated goat anti–rabbit IgG ( Sigma Chemical Co. ) or FITC-conjugated swine anti–rabbit IgG (DAKO Ltd.) was used to localize the Mok1 protein. A stock solution of latrunculin A (lat A; Molecular Probes Inc.) was made in DMSO (50 mM) and used at a concentration of 100 μM. DMSO alone did not affect the F-actin cytoskeleton or Mok1 distribution. Cells containing nmt1-mok1 + (SP103) grown at 29°C in minimal medium in the presence of 5 μM thiamine were filtered, resuspended in minimal medium without thiamine, and incubated for further 12 h to induce the nmt1 promoter. Then thiamine (5 μM) and cycloheximide (100 μg/ml) were added, cell extracts prepared every 15 min and immunoblotted with anti-Mok1 antibody. A genetic screen was performed in order to identify genes that regulate cell morphogenesis and function in the protein kinase C (PKC) pathway. Mutants that showed round or pear-shaped morphology at the restrictive temperature were visually isolated from a collection of ts or cs mutants. We have previously shown that two of the major targets of a protein kinase inhibitor staurosporine (STS) in fission yeast are PKC-related molecules, Pck1 and Pck2 , and that mutants defective in Pck1 and Pck2 function are supersensitive to this drug . Accordingly, the sensitivity of each round or pear-like mutant to STS was examined. In total 25 ts and 6 cs mutants were identified that satisfied both criteria. These loci were designated mok (morphological and kinase-inhibitor supersensitive). Complementation analysis of these mok mutants and known morphological mutants showed that they defined 10 different loci ( mok1-10 , Table II ). mok2 (eight alleles) and mok3 (one allele) were allelic to sts5/orb4 and pck2/ sts6 , respectively, which have previously been shown to produce STS-supersensitive round mutants . The most frequently isolated locus was mok1 (twelve ts and two cs alleles, Table II ). The localization of F-actin was examined to characterize the defective phenotypes of mok1 mutants. In fission yeast F-actin localizes to either the growing tips or the medial regions of dividing cells where the septum forms . In contrast to wild-type patterns, cortical actin did not show the specific localization in the mok1-664 mutant incubated at 35.5°C for 6 h. Instead, it localized in the cell cortex in a random punctate (right). Percentage of septated cells slightly increased at the restrictive temperature , suggesting that mok1 mutant is also defective in cell separation. The mok1 + gene is, therefore, required for the maintenance of rod-shape, cytokinesis and the cellular localization of F-actin. pck2 mutants are defective in cell wall integrity and, as a result, these cells are supersensitive to treatment with cell wall digesting enzymes . To determine the morphology defect of mok1 mutants was also due to an inability to maintain cell wall integrity, mutant cells were treated with β-glucanase. Like pck2 , the mok1 mutants were hypersensitive to β-glucanase even at the temperature permissive of 28°C . Defects in cell wall integrity can be sometimes compensated for by increases in the osmolarity of the growth media . Consistently mok1 mutant cells were capable of forming colonies at 35.5°C on rich YPD plates containing 1.2 M sorbitol . Also the mok1 mutants were hypersensitive to calcofluor, which disrupts cell wall architecture (data not shown). Therefore, the ts and morphological defective phenotypes of mok1 mutants were, at least in part, ascribable to defects in cell wall integrity. The mok1 + gene was cloned by complementation of the ts defect. Nucleotide sequencing of the cloned DNA fragment identified an uninterrupted ORF consisting of 7,230 bp which encoded a protein of 2,410 amino acid residues (the predicted molecular mass is 272 kD). Homology searching and structural analysis showed that the Mok1 protein comprises five structural domains . The first is the NH 2 -terminal 30 amino acid residues, which are highly hydrophobic and probably act as a signal peptide. The second domain follows the first 1,000 amino acid residues and has three transmembrane domains. A significant homology to a group of glycoside hydrolases, including bacterial α-amylase was found within this region . The third domain is a central, putative transmembrane, domain (amino acid residues 1,070– 1,090). The fourth comprises the next 1,000 amino acid residues which show significant homology to both bacterial glycogen synthase and plant starch synthase . Consistent with the homology between Mok1 and glucan synthases, two consensus sequences for UDP-glucose binding motifs were found in Mok1 . Mok1 and plant starch synthase further share a similarity in the N-terminal signal peptide and the region after the amylase-homologous region . The fifth domain is the COOH-terminal 400 amino acid residues that predict 12 membrane spanning domains . Thus the predicted structure suggests that Mok1 is an integral membrane protein that plays a role in degradation or synthesis of cell wall components. Homology searching against the fission yeast genome database (Sanger Centre) indicated that mok1 + constitutes a gene family; four putative homologues (designated mok11 + , mok12 + , mok13 + , and mok14 + ) are found in the genome sequences that are currently available. The predicted mok11 + (COOH terminus is unsequenced), mok12 + and mok13 + gene products also encode larger proteins (1,204, 2,352, and 2,371 amino acid residues, respectively), whereas mok14 + encodes a smaller protein (1,369 amino acid residues) which lacks the NH 2 -terminal amylase-homologous region. The overall homology among these proteins is 40–50% identity (50–60% similarity; Table III ) and all of them share virtually identical hydrophobicity profiles. It is of note that budding yeast does not contain any proteins that share significant homology to Mok1. Different mok1 mutants alleles were transformed with the noncomplementing plasmid pMK1101 (containing amino acid residues 1–876) and transformants were streaked and incubated at 35.5°C. In all nine ts mok1 mutant alleles tested, Ts + integrants appeared at a frequency of ∼10 −3 . Thus, the mutation sites in these mok1 mutants are in the region of the NH 2 -terminal 876 amino acid residues. The mok1 + gene was disrupted by three different strategies (Materials and Methods). In each case, tetrad analysis indicated that two viable and two inviable spores were obtained and that viable spores produced Ura − colonies. Microscopic observation of inviable spores showed that most of the Δ mok1 spores (18 out of 20 spores) failed to germinate. Therefore, mok1 + is essential for cell viability and also is required for germination. To examine phenotypes arising from overexpression of mok1 + , three different plasmids, which contained the whole ORF, the NH 2 -terminal 1,236 amino acid residues, and the COOH-terminal 1,393 amino acid residues respectively, were constructed in a vector carrying the thiamine-repressible nmt1 promoter . In addition, the nmt1 promoter was integrated into the genome in front of the mok1 + gene initiator methionine (ATG). It was found that overexpression of the entire mok1 + gene ( nmt-mok1 + ), either episomal or integrated, was toxic . Viability dropped sharply upon induction of the nmt1 promoter . In contrast, overexpression of the NH 2 -terminal or COOH-terminal half did not affect viability . Cell morphology after induction of nmt1-mok1 + was examined. Two types of cell shape defects were found. One showed an asymmetrical shape in which one end of the cell swelled abnormally to produce a tadpole . The other was observed as pairs of divided cells associated side-by-side (12%), the cell wall of which appeared fragile and often lysed upon division. To examine the deposition of cell wall material and the localization of actin in mok1 + -overexpressing cells, cells were stained with DAPI, calcofluor and rhodamine-conjugated phalloidin. In tadpole cells, cortical F-actin localized exclusively in the swollen tips . Calcofluor-staining showed a huge accumulation of cell wall material in the swollen parts of the tadpole cells . Time-lapse microscopy was performed after gene induction in order to follow morphological changes leading to the tadpole phenotype. Either the new or old end became swollen (the new end is the end that is produced after cytokinesis; the old end is the one that has already existed in the previous cycle). In Fig. 4 c, asymmetrical cell swelling that took place in the new end (marked with arrows) is shown. This analysis suggested that overproduction of Mok1 inhibited the translocation of F-actin (and cell wall materials) from one end to the other. In contrast to the asymmetrical accumulation of actin in tadpole cells, side-by-side cells showed neither actin staining nor the accumulation of cell wall material . These phenotypes suggest that these cells died after lysis. Nuclear staining by DAPI showed that each cell had a nucleus, indicating that chromosome segregation had been completed. In summary, the loss of Mok1 protein resulted in randomization of F-actin, whereas its overproduction abrogated translocation of F-actin from one end of the cell to the other as well as an abnormal accumulation of cell wall material and the lysis of some cells after cell division. As Mok1 contains two separate regions that display homology to amylase and glucan synthase respectively, we were interested in measuring the level of cell wall components in mutants in which the activity of Mok1 was either down- or upregulated. For this purpose, the level of glucan in cell walls was measured through the incorporation of radioactive glucose in either ts mok1 mutants or cells in which mok1 + was overexpressed. As shown in Fig. 5 and Table IV , the level of glucan was significantly altered by manipulation of Mok1. Most remarkably, α-glucan levels in Mok1-overproducing cells were elevated more than threefold over compared with wild-type cells (344%). The level of galactomannan also showed a modest increase (202%), whereas the level of β-glucan decreased slightly (75%). On the other hand, incubation of ts mok1 mutants at the restrictive temperature resulted in a decrease in α-glucan level by 69%. In contrast, galactomannan and β-glucan levels were virtually unchanged. These results strongly suggested that Mok1 is an α-glucan synthase. Polyclonal anti-Mok1 antibodies were prepared in order to characterize the Mok1 protein (Materials and Methods). These antibodies detected a band ∼280 kD after immunoblotting of total cell extracts from wild-type cells . The intensity of this band increased in cells containing the mok1 + gene on a multicopy plasmid (lane 2) and further dramatically increased in a strain in which mok1 + was overexpressed by induction of the nmt1 promoter (lane 3). These data showed that the antibody specifically recognized the mok1 + gene product. Total cell extracts were fractionated and immunoblotted using anti-Mok1 antibody. Mok1 was insoluble when a low salt buffer was used for the preparation of whole cell extracts or when the buffer contained high salt (0.5 M, lanes 4 and 5). Treatment with a nonionic detergent solubilized ∼50% of Mok1 (lanes 6 and 7) and ionic detergent resulted in complete solubilization (lanes 8 and 9). Protein denaturation with 8 M urea also solubilized Mok1 (lanes 10 and 11). Next the half-life of the Mok1 protein was examined. After a short induction of the mok1 + gene from the nmt1 promoter in minimal medium without thiamine (derepressed), p280 mok1 levels were followed by immunoblotting after the addition of thiamine and cycloheximide to repress promoter and protein synthesis respectively. It was found that Mok1 is a stable protein, a half-life is >120 min . These results suggested that Mok1 is an stable integral membrane protein, which is consistent with the presence of several transmembrane domains . Immunofluorescence microscopy was performed using anti-Mok1 in order to determine the subcellular localization of Mok1. As shown in Fig. 7 a, Mok1 mainly localizes in two regions, cell tips and the medial region. This localization of Mok1 is very similar to that for F-actin . Next, cells were simultaneously double stained with anti-Mok1 and anti-actin antibodies. Anti-Mok1 antibodies stained the region of the cell that overlapped with, but not identical to, actin staining . In a newly born small cell , just after cytokinesis, Mok1 and actin localized still in the new end in which the septum had previously been formed. Then these two proteins moved to the old end . Upon NETO , both actin and Mok1 were seen at the new end and these two proteins existed hereafter at both ends. It is of note that, while actin mostly localized as a patched pattern, Mok1 localization at the tips was much more homogeneous and produced a uniform staining pattern . At mitosis, actin and Mok1 disappeared from cell tips, and upon entry into anaphase , both proteins localized in the medial region of the cell as ring-like structures, where septa would subsequently form. Finally in septating cells , actin and Mok1 colocalized with septa and during septation (g), Mok1 staining broadened and looked as if it was doubled in width. In contrast, actin also now looked duplicated but the staining was less uniform and patched structures were more obvious. Finally, just before cell separation , Mok1 localized as dense dot that was bounded by two sets of actin patches in the center of the two dividing cells. It should be noted that localization to dense dot upon cytokinesis has been observed in several actin binding proteins such as Cdc8 and Myo2 . In summary Mok1 closely localizes with actin, at the growing cell tips in interphase, the medial regions in mitosis and a central dense dot during the final stage of septation. Given the localization of actin and Mok1 to the similar regions of the cell, we were keen to determine whether the actin cytoskeleton was required for the localization of Mok1. To address this question, F-actin was depolymerized with latrunculin A , and the consequence for the cellular localization of Mok1 determined. As shown in Fig. 8 a, after 10 min upon lat A addition (middle), Mok1 localization to both the growing ends and the medial regions was abolished. Immunoblotting showed that the total level of Mok1 unchanged during lat A treatment . These results suggested that cortical actin is essential for the correct localization of Mok1. To disrupt the actin cytoskeleton by another approach, the ts profilin mutant cdc3 and the ts tropomyosin mutant cdc8 were incubated at the restrictive temperature to disassociate F-actin patches from the cell tips and medial regions. Under these conditions, the F-actin patches associated with the cell periphery and the actomyosin ring structure failed to form, resulting in mitosis without cytokinesis and the accumulation of multi-nucleated cells . Mok1, as in lat A–treated cells, dislocalized completely in these cells (middle). We conclude that the cellular localization of Mok1 is dependent on the integrity of the F-actin cytoskeleton. The localization of Mok1 was examined in the ts mok1 mutants. As shown in Fig. 9 a, the Mok1 protein was no longer localized to discrete regions of the cell, instead the whole cytoplasm was stained. It should be noted that the presence of patch-like actin dots indicated that actin polymers attached with the plasma membrane as in wild-type cells in mok1 mutant cells . This mislocalized pattern of Mok1 was reminiscent of that in cells in which the actin cytoskeleton had been disrupted . The level of the Mok1 protein was indistinguishable between wild-type and ts mok1 mutants grown at the restrictive temperature , indicating that the apparent lack of the specific localization of Mok1 was not due to reduced protein levels. These results suggested that Mok1 localization requires intact Mok1 activity and/or structure and that the ts Mok1 proteins fail to be transported to the plasma membrane, which leads to randomization of F-actin and cell shape defects. We sought to examine the functional relationship between Mok1 and Pck1/2. To this end, the cellular localization of Mok1 was examined in pck2 mutant. It was found that Mok1 became mislocalized in this mutant ; neither the cell tips nor the medial regions were stained with anti-Mok1 antibody, instead a dispersed pattern was observed. Immunoblotting showed that the amount of Mok1 did not alter between wild-type and pck2 mutant (lower). In contrast the localization of F-actin appeared not to be severely impaired as cortical actin existed at either the cell tips or the medial regions (upper right). Next the double mutants between ts mok1 and Δ pck2 were constructed. mok1-664 Δ pck2 was synthetically lethal at 30°C, a temperature at which either single mutant could grew . Synthetic lethality was not observed between mok1-664 and Δ pck1 , consistent with our previous result showing that pck2 + plays the major role . Although mok1-664 Δ pck2 managed to form colonies at 27°C, double mutant cells were round and spontaneously lysed , suggesting that cell wall integrity was substantially impaired. This was confirmed by glucanase treatment of the double mutants grown at 27°C, as this strain was lysed much faster than either wild-type or each single mutant alone . These data indicate that Pck2 is required for the specific localization of Mok1 and that Mok1 regulates cell wall integrity either in parallel with or downstream of Pck1/2. To distinguish between the possibilities that Mok1 acts in parallel with or downstream of the Pck1 and Pck2 pathway, the nmt1-mok1 + gene was overexpressed in Δ pck2 cells (chromosomally integrated, SKP170). If Mok1 is somehow regulated by Pck1 and Pck2, Δ pck2 cells should become resistant to simple overexpression of mok1 + as Mok1 activity is expected to be compromised in the absence of Pck2 function. On the other hand, if Mok1 and Pck2 (and Pck1) regulate cell wall integrity in a parallel manner, nmt1-mok1 + should be as toxic in Δ pck2 cells as in wild-type cells or it might even be more toxic. As shown in Fig. 11 a, Δ pck2 cells became insensitive to nmt1-mok1 + , they were capable of forming colonies in the derepressed conditions, whereas wild-type cells were not. Consistent with this, calcofluor staining showed that morphology of cells in which mok1 + was overexpressed was less abnormal in Δ pck2 than in wild-type; neither tadpole cells nor asymmetrical accumulation of cell wall materials were observed . The lack of toxicity of nmt1-mok1 + was not due to a reduction in the levels of Mok1 in the Δ pck2 background . The level of cell wall glucan was measured in Δ pck2 mutant or that containing nmt1-mok1 + . α-glucan level was decreased in Δ pck2 mutant . The level of α-glucan in Δ pck2 cells containing nmt1-mok1 + was elevated, but the increase was greatly compromised compared with a wild-type strain containing nmt1-mok1 (46% lower). The reduced level of α-glucan together with mislocalization of Mok1 might explain why Δ pck2 mutant is tolerant to Mok1 overproduction. Taken together, the data suggested that Mok1 acts downstream, either directly or indirectly, of Pck1 and Pck2. Three lines of evidence indicate that Mok1 is a fission yeast α-glucan synthase. The amino acid sequence of Mok1 shows high homology to glycogen or starch synthase, and contains the consensus sequence for UDP-glucose binding. UDP-glucose is used as a substrate for glucan synthase. Second, overexpression of mok1 + results in 3.5-fold increase, while mutation of the gene results in a 30% reduction in the amount of α-glucan. Third, no homologous proteins exist in budding yeast, whose cell wall lacks the α-glucan . During the preparation of this manuscript, we learned that Hochstenbach et al. have also isolated mok1 + , and concluded that it encodes an α-glucan synthase (the gene was designated ags1 + ). The cell cycle–dependent targeting of Mok1 to the growing tips, the medial region and contractile ring-like structures is consistent with the identification of Mok1 as α-glucan synthase. These regions correspond to the sites at which cell surface structures are being actively remodeled, and both synthesis and degradation of the cell wall glucan take place to ensure tip growth, septum formation and cell separation respectively. We propose that in fission yeast 1,3-β- d -glucan synthase and glucanases also colocalize at these sites. Previous and ongoing work has suggested that Rho1 and Pck1 and Pck2 (Sayers, L., K. Nakano, I. Mabuchi, S. Katayama, T. Toda, and P. Parker, unpublished results) are intimately associated with the actin cytoskeleton. It is intriguing that all the proteins that constitute a sequential effector pathway (Rho1-Pck1/2-Mok1) appear to colocalize. The molecular mechanisms underlying the integration of these multiple enzymatic systems at these sites remain to be addressed. What is the molecular basis of Mok1 association with the plasma membrane? The presence of a putative signal peptide in the NH 2 terminus suggests that Mok1 is first translocated into the endoplasmic reticulum via the secretory pathway. It would be logical to suppose that this transport is followed by translocation into the plasma membrane through the Golgi and vesicle fusion . The dependency of Mok1 localization upon the integrity of the F-actin cytoskeleton is consistent with this idea. Recent analysis shows that polarized delivery of secretory vesicles requires actin cables rather than cortical actin and Mok1 localization may be also dependent on actin cables. Then why would F-actin be required to maintain Mok1 localization? The total level of Mok1 remained unchanged upon F-actin disruption, suggesting that it is unlikely that protein instability leads to the loss of specific localization of Mok1. It is possible that Mok1 turns over rapidly at the cell tips or medial regions and that, in the absence of F-actin integrity, it accumulates at some stage of the secretory pathway. This situation is analogous to actin, which turns over rapidly and disassembles instantly upon lat A treatment . pck2 mutant shows the reduced level of cell wall α-glucan and is resistant to toxic overproduction of Mok1. This suggests that Pck2 positively regulates Mok1 activity. In addition, Pck2 is required for Mok1 localization at the specific sites. Mok1 and Pck1/2 do not interact in yeast two-hybrid methods (Katayama, S., and T. Toda, unpublished results). It is possible that Pck2 regulates Mok1 indirectly via some regulators. Alternatively Mok1 may be a direct substrate of Pck2 such that Pck2-dependent phosphorylation is essential for the cellular localization and enzymatic activities of Mok1. The predicted amino acid sequence of Mok1 contains thirty consensus phosphorylation sites for PKC. It might be significant that all these sites are found in the domain that shows a homology to glycogen synthase. Although we have shown that Mok1 could be a downstream target of Pck1 and Pck2, it may not be the sole molecule by which Pck1 and Pck2 regulate cell morphology. In sharp contrast to the resistance of pck2 mutants to Mok1 overproduction, overproduction of Pck2 is as toxic in the ts mok1 mutants as in wild-type . This suggests that Pck1 and Pck2 regulate cell wall integrity via some effectors in addition to Mok1. Candidates include β-glucan metabolic enzymes, synthases or glucanases. Thus, in cells in which pck2 + is overexpressed, the level of β- as well as α-glucan increases (Arellano, M., and P. Pérez, unpublished results). It is highly possible that Pck1 and Pck2 regulate both α-glucan and β-glucan levels to ensure a coordinated regulation of the levels of these two major cell wall components. The Mok1 protein consists of several putative transmembrane domains, three of which lie in the NH 2 -terminal amylase-homologous domains, one in the central region of the protein and twelve at the COOH terminus. Consistent with this, the protein behaved as a membrane-bound protein. COOH-terminal tagging by fusion with DNA sequences encoding either HA-peptide or GFP completely abolishes Mok1 function. We suspect that tagging blocks the correct localization of Mok1 by disrupting the function of the membrane-spanning domains. In this context, it is of interest that the Mok1 protein derived from a ts mok1-664 strain also failed to localize correctly. As with the other eight ts mok1 mutant alleles examined, the mutation site in mok1-664 is not in the COOH terminus, but rather in the NH 2 -terminal amylase-homologous domain. The precise role of this domain is unclear at present, but it may be required not only for reorganization of α-glucan via a transglycosylase activity as suggested but also for the specific localization of Mok1. Multiple domains, therefore, appear to be responsible for the plasma membrane-bound localization of Mok1, and this association appears to be essential for Mok1 function. Generally genes encoding glucan synthases appear to comprise multi-gene families. In addition to five members of the Mok1 family of α-glucan synthase, the fission yeast genome contains at least three 1,3-β- d -glucan synthase homologues (Katayama, S., and T. Toda, unpublished results). In budding yeast, three 1,3-β- d -glucan synthase-encoding genes and two for 1,6-β- d -glucan synthase-encoding genes have been identified . Furthermore there are three chitin synthase-encoding genes in budding yeast and six in Aspergillus nidulans . The biological significance of five Mok1-related proteins is currently unclear as mok1 + is by itself essential for cell viability. Simultaneous triple deletions of the other four homologues (Δ mok11 Δ mok12 Δ mok14 ) do not affect viability in normal media. It is possible that these homologues become important in certain growth conditions such as stress and different growth media. It would be of significant to know how these multiple α-glucan synthase homologues are functionally distinct and differentially regulated during mitotic and meiotic cycles. | Study | biomedical | en | 0.999998 |
10087263 | Standard techniques and media were used for growth and genetic manipulation of yeast . Unless otherwise indicated, yeast cells were grown at 30°C. The yeast strains used in this study are described in Table I . In general, deletion mutants were constructed by PCR-based gene disruption as described . ΔFar1 strains were constructed either by PCR-based gene disruption ( Δ-1 ) or with a knockout cassette ( Δ-2 ). This cassette contained the FAR1 ORF followed by 100 bp 3′ sequence with URA3Kl replacing all but the first 109 codons. Far1-H7 , a far1 allele with a truncated COOH terminus , was constructed by replacing codons 757–830 of FAR1 with a stop codon followed either by HIS5Sp or URA3Kl . Gene disruptions were confirmed by PCR and phenotype including mating defects with wild-type and enfeebled testers, mating pheromone growth arrest, and budding pattern. The Δbud1 cdc24-m1 double mutant was crossed with appropriate wild-type haploid and random spore analyses demonstrated that the inability to shmoo in the presence of pheromone specifically segregated with Δbud1 cdc24-m1 and this mating defect was observed in both mating types. A single HA epitope (amino acids YPYDVPDYA) was added to the NH 2 terminus of STE4 by PCR. HASTE4 (including 394 bp 5′ upstream of the ATG) was cloned into pRS406 and two-step gene replacement of STE4 was used to construct RAY910. Protein A–tagged Far1p and Ste4p strains were constructed by PCR-based gene replacement using pZZ-His5 as template and oligonucleotides with 60 nucleotides 5′ and 3′ of the termination codon. Myc epitope-tagged Cdc24p and cdc24-m1p strains were constructed by PCR-mediated gene replacement as described except the sequence encoding a triple myc tag (MEQKLISEEDL MEQKLISEEDL MEQKLISEEDL) was directly fused to the Cdc24p NH 2 terminus. Gene replacements were confirmed by PCR and expression of epitope-tagged proteins of the correct size by immunoblotting using either 12CA5 (anti-HA) mAB tissue culture supernatant at 1:40 dilution, anti-protein A mAb ( Sigma ) at 1:2,000 dilution, or anti-myc polyclonal serum (Santa Cruz) at 1:500 dilution followed by ECL ( Amersham ). Strains with tagged proteins mated with wild-type mating efficiencies and arrested growth normally in response to α-factor. Cdc24HAGFP was constructed by fusing an HA epitope followed by PacI, SphI, NotI, and SacII restriction sites to the COOH terminus of Cdc24p using PCR and p414Cdc24 as a template. This resulted in p414Cdc24HA which had the amino acids YPYDVPDYAGLIKHARPPPRG fused to the COOH terminus. Yeast enhanced green fluorescent protein followed by the ADH terminator was PCR amplified from pMK199 (a gift from E. Schiebel) with an oligonucleotide that added a PacI site at the 5′ end and a NotI site at the 3′ end. This PCR product was cloned into p414Cdc24HA using PacI and NotI sites resulting in Cdc24p followed by YPYDVPDYAGLIKGSGAGAGAGAGA fused to GFP followed by the ADH terminator (p414Cdc24HAGFP). p416GalHASte4 was constructed by cloning HASTE4 into pRS416 containing the Gal1/10 promoter. The ADE2 gene from pSP73Ade2 (cloned by PCR from genomic DNA with oligonucleotides that added an EcoRI site at the 3′ end and a XhoI site at the 5′ end) was released by digestion with EcoRI and BsrGI followed by blunting. This fragment was cloned into pRS425 in which the LEU2 gene had been removed by digestion with Tth111I and NaeI followed by blunting resulting in p2μA. TPI- STE18 (triose phosphate isomerase promoter) from p416TSte18 (pRS416 with TPI cloned into the SacII EagI sites and STE18 cloned into BamHI EcoRI sites) was cloned into the SacI EcoRI sites of p2μA resulting in p2μATSte18. An oligonucleotide encoding the GAL4 nuclear localization signal (NLS) MDKAELIPEPPKKKRKVEL followed by a NcoI restriction site was cloned into EagI BamHI sites of p2μATSte18 yielding p2μATNLSSte18. Subsequently, an oligonucleotide encoding an HA epitope tag was cloned into the NcoI BamHI sites resulting in the following NLS-HA sequence, MDKAELIPEPPKKKRKVELPWMYPYDVPDYA fused to the NH 2 terminus of Ste18p yielding p2μATNLSHASte18. An EcoRI SacI fragment of p2μATNLSHASte18 containing TPI-NLSHA- STE18 was then cloned into pRS413 resulting in p413TNLSHASte18. STE18 was removed from this vector by digestion with BamHI and EcoRI and replaced with the coding sequence of FAR1 from pGAD424Far1 (see below) yielding p413TNLSHAFar1. The coding sequences of the entire FAR1 ORF and far1-H7 were amplified by PCR from genomic DNA and cloned into pGAD424. SpeI PstI fragments of FAR1 and far1-H7 from pGAD424 plasmids were cloned into pMal-c2 ( New England Biolabs ) resulting in pMFar1 (amino acid residues 133–831) and pMFar1H7 (amino acid residues 133–756). pMFar1ΔN (amino acid residues 638–831) and pMFar1ΔC (amino acid residues 133– 297) were derived from pMFar1 by removal of a BamHI or HindIII fragment, respectively. pMFar1H7ΔN (amino acid residues 638–756) was derived from pMFar1H7 by removal of a BamHI fragment. GSTCdc24 is comprised of the NH 2 -terminal 472 amino acids of Cdc24p fused to GST as described . Two-hybrid interactions were tested by growth on SC-leu-trp-his as described . Identical results were obtained with at least three transformants. Expression of a LacZ reporter from Y187 derived two-hybrid strains was quantified by β-galactosidase assays . An EcoRI site was inserted by oligonucleotide-directed mutagenesis after amino acid 153 of Spa2p . This 153–amino acid Spa2p fragment was then cloned into pGAD424. PJ69-4A cdc24-m1 was constructed by PCR-mediated gene replacement as described and confirmed by PCR and mating defect phenotype. Three independent PJ69-4A cdc24-m1 strains were used for two-hybrid analyses. Because TRP1 is used to replace CDC24 with cdc24-m1 , STE4 cloned into the 2μ URA3 GAL4 DBD vector pGBDU-C1 was used in this strain. Diploid two-hybrid strains were constructed by transformation of either DBD fusions or AD fusions along with p2μAT and p413T plasmids into SFY526 or Y187 and crossing these strains. After two-hybrid assays, phenotypes (diploid state and sterility) of diploid and haploid deletion two-hybrid strains were confirmed. Expression of NLSHAFar1p and NLSHASte18p in two-hybrid strains were confirmed by analysis of yeast extracts using SDS-PAGE, immunoblotting, probing with 12CA5 mAb, and ECL visualization. RAY1254, RAY1258, RAY1260, and RAY1336 cells carrying p416GalHASte4 were grown to an OD 600 of 0.5 in SC-ura with 2% (wt/vol) raffinose, galactose was added to a final concentration of 2% (wt/vol) and the cultures grown for 4 h. All subsequent steps were carried out at 4°C. Cells were harvested by centrifugation, and lysed by agitation with glass beads in buffer A (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 40 μg/ml each of leupeptin, chymostatin, pepstatin A, aprotinin, and antipain) containing 0.1% Triton X-100. Before use IgG–Sepharose was cross-linked with dimethylpimelimidate . Cell extracts were clarified by two centrifugations (10,000 g for 10 min). Supernatants, which contained the majority of the tagged proteins, were incubated with 20 μl of IgG-Sepharose ( Pharmacia ) equilibrated in buffer A containing 0.1% Triton X-100 for 1 h. Resin was then washed four times with buffer A containing 0.1% Triton X-100 and Far1-protein A fusions were specifically eluted by incubation with 20 U of TEV-protease ( Boehringer Mannheim ) for 4 h at 16°C in the same buffer. Eluates were analyzed by SDS-PAGE and immunoblotting using polyclonal sera against myc and Far1p (a gift from M. Peter) at 1:1,000 dilution followed visualization with ECL. All purification steps were carried out at 4°C. MBP and GST fusion proteins were expressed in E . coli with MBPFar1 and MBPFar1-H7 bacteria grown at 30°C. Cells were resuspended in buffer B (PBS, 1 mM DTT, 0.1% Triton X-100), frozen in liquid N 2 and stored at −70°C. Cells were lysed by sonication in buffer B with 1 mM PMSF. Extracts were clarified by centrifugation (10,000 g for 10 min) and fusion proteins were isolated using glutathione-agarose ( Sigma ) or amylose resin ( New England Biolabs ). MBP fusion proteins were eluted with 10 mM maltose in buffer B and dialyzed against buffer C (50 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , 1 mM DTT, 10% [vol/vol] glycerol). Protein concentrations were determined by the Bradford method or by comparing intensities of bands on Coomassie stained SDS-PAGE gels with BSA ( Sigma ) as a standard. For both MBPFar1 and MBPFar1-H7, concentrations used refer to the full-length protein and not proteolytic breakdown products. HASte4-(TEV)-protein A was purified from RAY1276 cells using IgG-Sepharose under conditions similar to those described in . Cells were grown in YEPD to an OD 600 of ∼3, harvested by centrifugation, resuspended in 20 mM Tris-HCl pH 7.4 with 50 mM NaCl at ∼300 OD 600 /ml, snap frozen in liquid N 2 , and stored at −70°C. Typically 2,500 OD 600 of cells were broken in buffer D (buffer A containing 2 mM EDTA and 3 mM MgCl 2 ) by agitation with glass beads. Triton X-100 was added to cell extracts at a final concentration of 1%. After 1 h incubation the extract was centrifuged at 10,000 g for 20 min. The supernatant was incubated overnight with 250 μl IgG-Sepharose equilibrated in buffer D containing 1% Triton X-100. The resin was collected by centrifugation, washed once with buffer D containing 1% Triton X-100 and twice with buffer D containing 0.1% Triton X-100. HASte4p was specifically eluted by incubation with 20 U of TEV-protease in 400 μl buffer D containing 0.1% Triton X-100 for 5 h. Comparison of the amounts of total protein and HASte4p in yeast extracts (treated with TEV-protease) and eluted HASte4p preparations indicated that HASte4p was enriched over 1,000-fold in comparison to cell extracts. By immunoblotting both 3xmycCdc24p and Far1p were undetectable in HASte4p preparations (<0.01% of the starting level). Binding experiments were all carried out at 4°C. For binding of GSTCdc24 and MBP fusion proteins ∼10 μg of GSTCdc24 bound to glutathione-agarose was incubated with respective MBP fusion proteins in 100 μl of buffer C overnight. Glutathione-agarose samples were washed twice with 1 ml of buffer C and once with 1 ml of buffer B. Proteins were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Coomassie blue staining or transfer to nitrocellulose, probing with anti-MBP mAb ( Sigma ) at 1:4,000 dilution and visualized by ECL. For binding experiments with yeast HASte4p, the HASte4p preparation was diluted 10-fold into buffer C and 100 μl was incubated with either resin bound GST or MBP fusions. MBP fusions were bound to amylose resin by incubation of ∼5 μg of each protein with 20 μl of amylose resin for 1 h. GSTCdc24–MBPFar1 was prepared by passing a bacterial extract (from 100 ml of cells) containing GSTCdc24 over a column with ∼500 μg of MBPFar1 bound to amylose resin. The column was washed with buffer B and then GSTCdc24–MBPFar1 was eluted with buffer B containing 10 mM maltose. The eluate was incubated with glutathione-agarose for 30 min which was then washed three times with buffer B. Proteins bound to the resin were analyzed by SDS-PAGE and Coomassie blue staining or used for HASte4p binding. Quantitative matings were carried out as described in with Mat a cells as indicated and Matα RAY1135 cells. Pheromone induced cell cycle arrest and induction of a Fus1LacZ reporter were assayed as described . For pheromone treatment ∼0.2 OD 600 of log-phase cells were collected by centrifugation, resuspended in 2 ml YEPD containing 12 μM α-factor (synthesized by David Owen, MRC LMB) and incubated for 3 h. Cells were fixed with formaldehyde and actin was visualized as described using rhodamine phalloidin (Molecular Probes). To examine cell morphologies in mating mixtures, Mat a cells were stained with 10 μg/ml Calcofluor white ( Sigma ) in YEPD for 5 min at rt and subsequently washed extensively with YEPD. Approximately 5 × 10 6 stained cells were then mixed with unstained Mat a cells and incubated on filters. After 2 h cells were washed from the filters, briefly sonicated, resuspended in PBS and fixed with formaldehyde. Images of cells were taken using a Zeiss Axioskop microscope with either a NA 1.4 ×63 or NA 1.3 ×100 objective and recorded with a Princeton Instrument Micromax CCD camera. Fluorescence and differential interference-contrast (DIC) images were merged to permit identification of Mat a cells. Cdc24HAGFP (p414Cdc24HAGFP) was transformed into RAY931 which is deleted for CDC24 and kept alive by the rescuing plasmid pEG(KT)Cdc24 . This strain was able to lose the rescuing plasmid (both by extensive growth in SC-trp media or counter-selection on 5-FOA) as determined by markers and PCR, resulting in RAY1360, indicating that Cdc24HAGFP was functional. RAY1360 grew normally at 22°C, 30°C, and 37°C on YEPD plates. Budding patterns were determined as described and mating efficiency was determined as described above. Cdc24HAGFP expression and size was verified by SDS-PAGE, immunoblotting, probing with 12CA5 mAb and ECL visualization. Confocal microscopy was carried out as described except cells were grown in SC supplemented with 55 μg/ml adenine to reduce fluorescence due to ade2 . Pheromone treatment was with 140 μM α-factor. Cells were imaged after 1 h in order to observe early localization. For latrunculin A treatment of budding cells 2 μl of either 10 mM latrunculin A (Molecular Probes) in DMSO or DMSO was added to 200 μl of log-phase cells (final concentration latrunculin A 0.1 mM) and cells were incubated for 3 h . For latrunculin A treatment of shmoos, cells were incubated with 140 μM α-factor for 1 h and then 0.1 mM latrunculin A or DMSO was added to cells which were incubated for 2 h. After observation by confocal microscopy, actin depolymerization was confirmed by staining fixed cells with rhodamine phalloidin as described above. Cdc24-m alleles are defective in growth orientation along a pheromone gradient, yet do not affect pheromone-dependent MAP-kinase pathway signaling . These mutants are also unable to interact with Gβ (Ste4p) in two-hybrid assays. In Cdc24p–Gβ two-hybrid assays Gβ was overexpressed, which has been shown to activate the MAP-kinase pathway . Hence it was possible that MAP-kinase signaling is required for this interaction. Two-hybrid experiments revealed that while Cdc24p and Gβ interact in a haploid strain, no detectable interaction was observed in a diploid , in which several mating specific proteins, including Gγ (Ste18p), are not expressed . Gγ is required for this interaction in a haploid . Surprisingly, overexpression of Gγ in a diploid did not restore the Cdc24p–Gβ interaction, whereas overexpression of Gγ in a Δste18 haploid restored this interaction (Table II ). This result is consistent with the notion that either a haploid specific component and/or pheromone-dependent signaling is required for the Cdc24p–Gβγ interaction. To examine the role of the pheromone-dependent MAP- kinase pathway in the Cdc24p–Gβγ interaction, two-hybrid strains were constructed in which each component of this pathway was deleted. The MAP-kinase scaffolding protein Ste5p, the PAK kinase Ste20p which phosphorylates Ste11p, the MAPKKK Ste11p, the MAPKK Ste7p, the MAPK Fus3p or Kss1p, and the transcription factor Ste12p were each individually disrupted in a two-hybrid strain. In addition, the Ste20p homologue Skm1p, the cyclin-dependent kinase inhibitor required for α-factor cell cycle arrest Far1p, the polarity establishment protein Bem1p, the Gβγ effector Akr1p, and the bipolar bud site selection protein Bud6p were deleted from this strain. Several of these proteins including Ste5p, Fus3p, and Far1p are only expressed in haploids and thus are candidates for haploid specific components required for the Cdc24p–Gβγ interaction. Deletion of SKM1 , BEM1 , AKR1 , or BUD6 had no effect on the Cdc24p–Gβ interaction. In contrast, removal of any component of the pheromone-dependent MAP-kinase pathway (with the exception of Fus3p and Kss1p which are functionally redundant for mating) resulted in the loss of the Cdc24p–Gβ two-hybrid interaction. Because Gγ appeared to be required for the Cdc24p–Gβ interaction , we examined whether overexpression of Gγ was able to restore the Cdc24p–Gβ interaction in these strains. Table II shows that overexpression of Gγ partially restored the Cdc24p–Gβ interaction in Δste5 , Δste11 , and Δste7 strains and to a lesser extent in Δste20 and Δste12 strains. These results indicate that signaling through the pheromone-dependent MAP-kinase cascade per se is not required for the Cdc24p–Gβγ interaction. However, deletion of FAR1 resulted in a loss of the Cdc24p–Gβ interaction which was not restored upon overexpression of Gγ , suggesting that Far1p may be essential for this interaction. The requirement for Gγ and Far1p in the Cdc24p–Gβ two-hybrid interaction suggested an explanation for the low level of LacZ reporter activity observed in the haploid Y187 two-hybrid strain and the absence of an interaction in the diploid two-hybrid strain, namely that these two proteins were limiting in haploids and absent in diploids. To test this possibility, we overexpressed Gγ and Far1p individually and together in the Y187 haploid two-hybrid strain. Fig. 1 A shows that overexpression of Gγ in the presence of pAS1Cdc24 and pGAD424Ste4 resulted in an approximately twofold increase in LacZ activity, whereas the additional overexpression of Far1p resulted in a further increase in LacZ activity by ∼3.5-fold. In diploids, overexpression of Gγ did not result in a Cdc24p–Gβ interaction. However overexpression of Far1p resulted in LacZ reporter activity and this was further increased by additional overexpression of Gγ, suggesting that in the absence of pheromone-dependent signaling Far1p is sufficient for restoring the Cdc24p–Gβγ interaction. FAR1 is necessary for both pheromone-dependent growth arrest and oriented growth towards a pheromone gradient . These two functions of FAR1 can be separated, with the Far1p NH 2 terminus necessary for cell cycle arrest and the COOH terminus necessary for growth orientation. Our two-hybrid results indicate FAR1 is necessary for the Cdc24p–Gβ interaction, yet it is unclear which function of FAR1 this corresponds to. Because cdc24-m and far1-s appear phenotypically identical and both exhibit orientation defects , we examined the effect of the far1-s allele far1-H7 on this interaction. This far1 mutation results in a COOH-terminal 75– amino acid deletion and despite its orientation defect is normal for cell cycle arrest. Fig. 1 C shows that a far1-H7 mutation prevents the Cdc24p–Gβ interaction. These results suggest that the FAR1 orientation function is required for the Cdc24p–Gβ association, consistent with the role of this interaction in growth orientation. We next investigated whether Far1p interacted with Cdc24p and Gβ. Fig. 1 and Table III show that in two-hybrid assays Far1p can interact with both Cdc24p and Gβ. The Cdc24p–Far1p interaction was observed in strains deleted for STE4 , STE18 , FUS3 , or STE12 , indicating that it does not require Gβγ nor pheromone-dependent MAP-kinase signaling (including Fus3p-dependent phosphorylation of Far1p). Similarly, the Far1p–Gβ interaction did not require STE18 , FUS3 , or STE12 . The Far1p–Gβ interaction also did not require the CDC24 orientation function as we observed this interaction in a cdc24-m1 two-hybrid strain. In addition we examined Cdc24-m1p, which we had previously shown does not interact with Gβ, and found that Cdc24-m1p also did not interact with Far1p (data not shown). Together these results suggest that Far1p and Cdc24p can associate and this association is independent of pheromone signaling. To further investigate these interactions epitope-tagged versions of Far1p and Cdc24p were constructed. Myc and protein A domains were fused to Cdc24p and Far1p, respectively and these fusions were used to replace wild-type genes. Far1-protein A fusions had a tobacco etch virus (TEV) protease cleavage site between Far1p and protein A to allow specific elution. These strains grew normally and exhibited normal vegetative morphology. Furthermore, both fusions mated with similar efficiencies as a wild-type strain when crossed to a wild-type tester or to an enfeebled tester. Far1-protein A fusions promoted normal cell cycle arrest and cells carrying this fusion formed shmoos that appeared normal upon exposure to mating pheromone. Together these results indicated that the fusion proteins were functional. Fig. 2 shows that when Far1-protein A was isolated with IgG-Sepharose, myc-tagged Cdc24p was bound (compare lanes 1 and 2 with 3 and 4). When Cdc24p or Far1p orientation mutants were used, a substantial decrease in the amount of Cdc24p bound to Far1p was observed in both cases (lanes 5–8). These results reveal the molecular basis for the similar phenotypes of cdc24-m1 and far1-H7 mutants. Although two-hybrid results indicated a Far1p–Gβ association, this was apparently not stable enough to observe by immunoprecipitation. To address whether these protein interactions were direct, binding experiments were carried out using purified proteins. Far1p and the NH 2 -terminal half of Cdc24p were purified from bacteria as fusions to maltose binding protein (MBP) and glutathione- S -transferase (GST), respectively. Fig. 3 A shows that MBPFar1 bound GSTCdc24 but not GST alone. MBPFar1-H7 does not significantly bind GSTCdc24p, consistent with immunoprecipitation results above . In these binding experiments an excess of GSTCdc24 is used and an increase in MBPFar1 binding occurs as its concentration in the binding reaction is increased . These results demonstrate that Far1p can bind Cdc24p directly in the absence of other proteins. These binding studies demonstrated that the COOH terminus of Far1p is necessary for GSTCdc24 binding, hence we examined if this region was also sufficient for binding. Fig. 4 B shows that a 200–amino acid Far1p COOH-terminal fragment (lane 10) is not sufficient for GSTCdc24 binding and furthermore an NH 2 -terminal Far1p fragment did not bind GSTCdc24 (lane 9). MBPFar1-H7 and MBPFar1ΔC, which do not bind GSTCdc24 are unlikely to be grossly misfolded as they retain the ability to bind Gβ (see below). These results indicate that although the COOH terminus of Far1p is necessary for binding Cdc24p it is not sufficient. An immunoblot of the MBPFar1 bound to GSTCdc24 revealed that proteolytic fragments of Far1 with as little as 25 kD of the NH 2 -terminus (approximately residues 133–350) are coprecipitated with MBPFar1 and GSTCdc24. This region of Far1p, which includes a Lim domain , does not bind Cdc24p directly , suggesting that this region may mediate Far1p multimerization. Hemagglutinin (HA)-tagged Gβ (Ste4p) was purified from yeast in order to examine its binding to MBPFar1 . For this purpose a strain in which the wild-type copy of STE4 was replaced with HASte4-(TEV)-protein A was used. This fusion was functional for mating and cell cycle arrest. The Ste4p fusion was isolated with IgG-Sepharose and eluted by specific cleavage between Ste4p and the protein A domains using TEV protease, yielding HASte4p which was over 1,000-fold enriched compared with cell extracts. This HASte4p preparation had undetectable levels of Far1p and Cdc24p (<0.01% of the starting level, data not shown). Aliquots of HASte4p were incubated with various MBPFar1 fragments immobilized on amylose resin and bound HASte4p was analyzed by SDS-PAGE and immunoblotting. Fig. 4 C shows that HASte4p bound equally well to MBPFar1 and MBPFar1-H7 (lanes 2 and 3), whereas both NH 2 - and COOH-terminal Far1p fragments bound substantially less HASte4p (lanes 4–6). Of these smaller Far1p fragments, only Far1ΔC (amino acid residues 133–297) bound substantial amounts of HASte4p, suggesting that this region which includes the Lim domain is involved in Gβ binding. These experiments show that Gβ can bind Far1p in the absence of Cdc24p and suggest that perhaps Gβ and Cdc24p bind to different regions of Far1p. Since both Cdc24p and Far1p bind Gβ, we addressed whether the addition of MBPFar1 to GSTCdc24 bound to glutathione-agarose could compete for HASte4p (Gβ) binding. Fig. 5 A shows that addition of MBPFar1 did not prevent HASte4p binding to GSTCdc24 (compare lanes 1 and 2 with 3 and 4), but rather increased binding by about twofold. Coomassie blue staining of glutathione-agarose eluates revealed that MBPFar1 bound GSTCdc24. These results indicate that Far1p binding to Cdc24p does not displace Gβ, and are consistent with the formation of a complex of all three proteins. To directly test whether a complex of all three proteins could form we determined whether a stoichiometric complex of Cdc24p–Far1p could bind Gβ (Ste4p). GSTCdc24– MBPFar1 was isolated by sequential purification using amylose and glutathione resin. Fig. 5 B shows that GSTCdc24–MBPFar1 contained roughly equal amounts of these two fusion proteins. Purified HASte4p was then incubated either with this complex or GSTCdc24 alone. Densitometric quantification showed that twofold more HASte4p bound to Cdc24p–Far1p than to a similar amount of Cdc24p alone, demonstrating that trimeric Cdc24p-Far1p-Gβ can form. This increase in Gβ binding does not appear to be cooperative and is more likely to be the sum of contributions from Cdc24p and Far1p. Because both Far1p and Cdc24p can individually bind each other or Gβ it is likely that in a trimeric complex each protein contacts the other two proteins. These binding studies together with the two-hybrid results suggest that Cdc24p-Far1p-Gβ is necessary for mating projection orientation. To examine if CDC24 and FAR1 function in the same process we compared the mating efficiencies of both single and double Δfar1 and cdc24-m1 mutants. Fig. 6 shows that the presence of a cdc24-m1 mutation in a Δfar1 background did not result in a further decrease in mating efficiency, suggesting that FAR1 and CDC24 function in the same orientation process. The mating defect of the double mutant is closer to that of the Δfar1 mutant that in addition to a chemotropism defect does not arrest growth in response to mating pheromone. If cdc24-m1 affected chemotropism similarly to Δfar1 , then a double mutant with Δspa2 , a gene required for the default mating pathway , should have a mating defect greater than the product of the individual mating defects, a phenomenon known as synthetic sterility . Fig. 6 shows that Δspa2 cdc24-m1 mutants exhibited synthetic sterility. We also examined genetic interactions between cdc24 - m1 and Δbem1 or Δste20 , two genes involved in polarized growth and mating. Bem1p is associated with the cytoskeleton , binds Cdc24p , and Far1p . Bem1 mutants are unable to form shmoos and instead form round cells in the presence of mating pheromone . ΔBem1 cdc24-m1 cells showed similar temperature sensitive growth and morphological defects (large round cells) as Δbem1 cells, providing further evidence that cdc24-m1 has no effect on vegetative growth. Even in cells lacking BEM1 which cannot form shmoos, cdc24-m1 resulted in a substantial decrease in mating efficiency , i.e., synthetic sterility. Because deletion of the PAK kinase STE20 in our strain background did not result in complete sterility, we were able to examine the mating defect of Δste20 cells in the presence and absence cdc24-m1 . In the absence of STE20 , cdc24-m1 resulted in a further decrease in mating efficiency. Together these results suggest that in Δbem1 and Δste20 mutants, which are unable to form shmoos, polarization may still be necessary for mating perhaps for the localization of proteins necessary for cell fusion. Furthermore, because BEM1 and STE20 are not required for default mating , such synthetic mating defects with cdc24-m1 are consistent with a genetic linkage between shmoo formation and orientation. If Cdc24p transmits signals from bud site selection proteins or Gβγ, it might be localized to regions of polarized growth. We therefore examined the localization of a Cdc24p green fluorescent protein (GFP) fusion. Cdc24HAGFP expressed from its own promoter on a CEN plasmid complemented Δcdc24 as determined by growth at different temperatures, budding patterns, and mating efficiencies (data not shown). Fig. 7 A shows the localization of Cdc24HAGFP in living cells at different stages in the cell cycle. In unbudded cells Cdc24p localized as a tight patch at the membrane, and in cells with small buds at the growing end. In larger buds, this localization became more spread out. Finally, during cytokinesis Cdc24p generally localized to the mother-bud neck. Curiously, a preliminary report showed that an overexpressed GSTCdc24 fusion protein had a circumcellular distribution in budding cells . Cdc24HAGFP also localized to sites of polarized growth after α-factor treatment. Fig. 7 B shows different shmoos in which Cdc24HAGFP is observed as a patch at the tip of the mating projection. Cdc24HAGFP was localized similarly in mating mixtures (data not shown). Furthermore, as the sole copy of Cdc24p in a Δcdc24 strain, Cdc24-m1HAGFP also localized to sites of polarized growth in budding and mating cells (data not shown), indicating that this mutant is not defective in its localization to sites of polarized growth. These data demonstrate that Cdc24p localizes to sites of polarized growth. The early localization of Cdc24p in the cell cycle and its localization to the shmoo tip are consistent with its function in polarity establishment. The localization of Cdc24p is similar to that of its substrate Cdc42p . To determine whether the actin cytoskeleton was necessary for polarized Cdc24p localization, budding and shmooing cells were treated with the actin depolymerizing drug latrunculin A . Fig. 8 A shows that even in the absence of actin polymerization, Cdc24p is localized to sites of polarized growth in budding cells. In contrast, latrunculin A treatment of shmoos resulted in a substantial decrease in Cdc24p localization . Upon latrunculin A treatment the number of cells with Cdc24p localized to the shmoo tip decreased by fivefold ( n = 100) and in cells that exhibited localized Cdc24p, there appeared to be a decrease in the amount of Cdc24HAGFP localized to the shmoo tip and an increase in fluorescence throughout the cell. These results are consistent with the effects of latrunculin A on Cdc42p localization in budding and shmooing cells . During mating, a pheromone gradient serves as the external cue for growth orientation. This external signal allows haploid cells to orient growth in a pheromone gradient emanating from any direction , whereas the site for bud formation in haploids is fixed adjacent to the previous bud site . The selection of a site for the mating projection must override the fixed location of the bud. If Cdc24p acts as a switch between internal signals during budding and external signals during mating , we would predict that bud site selection proteins become important for cell mating when the capacity for shmoo orientation is lost in mutants such as cdc24-m1 . The ras related small G-protein Bud1p/Rsr1p is essential for bud site selection, yet is not required for chemotropic or default mating in saturating pheromone . However Bud1p can directly associate with Cdc24p and this association is likely to be functionally important . We therefore examined the phenotype of Δbud1 cdc24-m1 double mutants to determine if the loss of CDC24 -mediated chemotropism caused a BUD1 -dependent mating defect. Both Δbud1 and Δbud1 cdc24-m1 cells grew normally, were not temperature sensitive for growth, and had the expected random budding pattern (data not shown). Strikingly, the Δbud1 cdc24-m1 double mutant showed a stronger mating defect (an eightfold further decrease in mating efficiency) than cdc24-m1 alone . In contrast, Δbud1 alone had no effect on mating efficiency in agreement with previous studies . Microscopic observation of Δbud1 cdc24-m1 double mutants treated with a high concentration of mating pheromone or exposed to pheromone gradients in mating mixtures revealed that these cells were defective in shmoo formation. Instead of forming typical pear-shaped shmoos most cells were enlarged and round. On closer inspection a small protrusion was occasionally observed on these cells. Furthermore, the actin cytoskeleton in the double mutants was depolarized, with actin cortical patches and cables disorganized . In contrast, both Δbud1 and cdc24-m1 single mutants formed shmoos. Otherwise Δbud1 cdc24-m1 double mutants responded normally to pheromone by undergoing cell cycle arrest and pheromone-dependent gene induction (data not shown). These results suggest that in the absence of chemotropism, BUD1 and perhaps the bud site selection machinery becomes essential for shmoo formation. Surprisingly, in saturating uniform concentrations of mating pheromone Δbud1 does not result in a mating defect , raising the possibility that this novel role of BUD1 is revealed specifically when signaling from Gβγ to Cdc24p is blocked. Our results indicate that Far1p is required for signaling from Gβγ to Cdc24p. If the shmoo formation defect of Δbud1 cdc24-m1 cells is due to a defect in this signaling, a Δbud1 Δfar1 double mutant should show an analogous decrease in mating efficiency. ΔBud1 Δfar1 cells had a stronger mating defect (an eightfold decrease in mating efficiency) than Δfar1 cells. As a control the effect of Δbud1 was examined in a Δsst2 strain. ΔSst2 cells are supersensitive to mating pheromone as SST2 negatively regulates the heterotrimeric G-protein . Therefore Δsst2 cells mate as though they are saturated with mating pheromone, mating by the default pathway . ΔBud1 Δsst2 cells had a similar mating defect as Δsst2 alone, indicating that the absence of chemotropic mating by itself is not sufficient to reveal BUD1 function in mating. These synthetic mating defects of Δbud1 with cdc24-m1 or far1 show that Bud1p, which normally functions in bud site selection, can play a role in shmoo formation, presumably by regulating Cdc24p. During mating yeast cells grow in a polarized fashion towards their mating partner . Yeast cells are able to sense pheromone gradients and orient their actin cytoskeleton and secretion towards such a gradient. Here we show that a complex comprised of Cdc24p, Far1p, and Gβγ can form and is likely to be required for orientation towards a mating partner. The formation of this complex does not directly require signaling via the pheromone-dependent MAP-kinase pathway. Analyses of mating defects of double mutants indicate that FAR1 and CDC24 both function in the same cell orientation process. Cdc24p localizes to sites of polarized growth suggesting that Cdc24p-Far1p-Gβγ is localized. Cdc24p localization does not depend on the actin cytoskeleton during budding but does depend on the actin cytoskeleton during shmooing. In the absence of signaling from Gβγ to Cdc24p, the bud site selection protein Bud1p is required for shmoo formation, demonstrating a molecular link between growth site selection in mating and budding. Together these results suggest that binding of Gβγ to Far1p and Cdc24p creates an internal landmark for growth towards an external signal. Detection of a pheromone gradient and orientation of growth in such a gradient is a process central to yeast mating and is analogous to Dictyostelium chemotaxis and nerve cell chemotropism . Alleles of both far1 and cdc24 are specifically defective in orientation towards a pheromone gradient. Cells mutant for the α-factor pheromone receptor (Ste2p) or the heterotrimeric G-protein, discriminate poorly between pheromone signaling and nonsignaling mating partners suggesting that these components are also required for chemotropism . Cdc24 - m mutants are unable to interact with the Gβ subunit of the heterotrimeric G-protein . These results led to a model in which Gβγ locally activates or recruits Cdc24p, which could then activate Cdc42p and other downstream targets required for cytoskeleton orientation. We conclude from two-hybrid, binding, and genetic data that Far1p is involved in signaling from Gβγ to Cdc24p by forming a complex with these proteins. Our two-hybrid results suggest that the Far1p–Gβ interaction does not require the CDC24 orientation function, yet Far1p is essential for the Cdc24p–Gβ interaction. In contrast, in vitro binding experiments show that Cdc24p is able to bind to Gβ purified from bacteria and yeast in the absence of Far1p. We attribute this difference between two-hybrid and in vitro binding results to the different methods used. For example, in the two-hybrid experiments interactions occur in the nucleus and on the other hand the in vitro binding studies are carried out with high concentrations of purified proteins. We suggest that although Far1p is important for the Cdc24p–Gβ interaction it is not absolutely essential, whereas Cdc24p is not necessary for the Far1p–Gβ interaction. We have demonstrated that a triple complex comprised of Cdc24p-Far1p-Gβγ can form using purified proteins and believe that in vivo this complex links receptor activation to cytoskeleton organization . These results show at a molecular level the role of Far1p in growth orientation. Consistent with the specific phenotype of far1 and cdc24 orientation alleles, we find that pheromone-dependent MAP-kinase cascade signaling is not necessary for the association of this complex. This result indicates that the MAP-kinase cascade is not directly required for chemotropic growth, in agreement with recent mating partner discrimination studies . Furthermore, the formation of this complex does not require FUS3 , which normally phosphorylates Far1p in a pheromone-dependent fashion . This phosphorylation of Far1p is necessary for cell cycle arrest, indicating that the cell cycle arrest function of FAR1 is not required for interactions between Cdc24p, Far1p, and Gβγ. How is the formation of this protein complex regulated by pheromone activation of the receptor? Pheromone binding to the receptor is believed to trigger dissociation of Gα from Gβγ. Recent studies suggest Gα binds the pheromone receptor and that Gβγ must be membrane associated in order to function . GFP fused to Gγ is localized preferentially to the plasma membrane of the mating projection after pheromone treatment (Nern and Arkowitz, unpublished observation). Upon pheromone stimulation, Far1p levels increase resulting in increased levels of Cdc24p–Far1p (Nern and Arkowitz, unpublished observation). As Far1p localizes to the nucleus during vegetative growth , it would appear likely that Far1p must exit the nucleus in order to carry out its mating orientation function. We envision that released Gβγ recruits Cdc24p–Far1p to the vicinity of activated receptors and Cdc24p-Far1p-Gβγ ultimately directs the cytoskeleton towards this internal landmark. Such a mechanism provides a means of translating local activation of pheromone receptors to cytoskeletal orientation. We would predict that Far1p, like Cdc24p, localizes to the tip of the mating projection in pheromone treated cells. While this work was being reviewed a paper examining the role of Far1p in polarized growth during mating was published . In general, our results agree with the findings of this work. The authors postulate that Far1p functions as an adaptor or linker between Gβγ and polarity establishment proteins including Cdc24p. Our in vitro binding results indicate that even in the absence of Far1p, Gβγ can still bind Cdc24p, suggesting that perhaps Far1p is not simply a physical adaptor but may have more complex functions. Overexpressed GFPFar1 was shown to relocalize from the nucleus to the cytoplasm upon treatment with a saturating uniform concentration of pheromone for two hours. In these conditions GFPFar1 does not appear to accumulate at shmoo tips. It will be interesting to determine whether wild-type levels of Far1p localizes similarly in cells exposed to a pheromone gradient for various times. How are these protein interactions involved in transmitting spatial information? Previous studies have indicated that in a pheromone gradient, shmoo orientation improves as a function of time . This appears to be due to reorientation of the shmoo tip as it grows , indicating that shmoo orientation is a continuous process unlike bud site selection. Perhaps Cdc24p-Far1p-Gβγ dissociates reasonably fast such that this complex is continually dissociating and forming. Such a dynamic process would provide a means for continuous reorientation during mating and could play a central role in translating initial small differences in receptor occupancy into oriented growth. Another difference between bud and shmoo formation is that in budding the polarity establishment proteins Cdc42p and Bem1p localize independent of the actin cytoskeleton whereas in the latter process the actin cytoskeleton is necessary for the efficient localization of these proteins . During shmoo formation the actin cytoskeleton requirement for localization of Cdc24p and these other polarity establishment proteins appears to be similar. Why is the actin requirement for localization of this group of proteins different in budding and shmooing cells? Perhaps the continuous nature of the shmooing process compared with the committed directional growth required for budding underlies this different dependence on the actin cytoskeleton. It will be important to examine the role of the actin cytoskeleton in cells responding to a pheromone gradient. Pheromone stimulation results in gene induction, cell cycle arrest, and morphological changes . The timing and coordination of these different responses is important for efficient mating. Our genetic studies are consistent with Cdc24p and Far1p being part of the same protein complex functioning in growth orientation and we examined two additional genes that might have a role in coordinating various pheromone responses. The PAK kinase Ste20p is important for MAP-kinase signaling during mating. It interacts with Bem1p, Ste4p, Ste5p, and Cdc42p . Recent mating partner discrimination studies suggest that STE20 may not be required for chemotropism. Our two-hybrid results suggest that STE20 has some effect on the Cdc24p–Gβ interaction, however cdc24- m1 results in a further mating defect in Δste20 cells. While Ste20p binds Gβ, it is unclear how this association relates to the Far1p, Cdc24p, Gβγ interaction. Further studies will be necessary to elucidate the roles of STE20 in various aspects of mating. Bem1p is required for polarized growth both during mating and budding . ΔBem1 cells are defective in shmoo formation, mating pheromone-dependent cell cycle arrest and efficient signaling via the MAP-kinase cascade . At the molecular level Bem1p interacts with many components required for polarized growth such as the G-protein Bud1p , Cdc24p , Far1p , actin , Ste5p , and Ste20p . Although Bem1p binds both Cdc24p and Far1p, Bem1p is not required for the formation of Cdc24p-Far1p-Gβγ. Results from Δbem1 cdc24-m1 mutants suggest that even for cells unable to form shmoos, polarization is important. Perhaps this is because the molecules necessary for cell fusion must be correctly localized. What is the molecular function of Bem1p in mating? We favor the idea that Bem1p acts as a scaffolding component linking pheromone-dependent MAP-kinase signaling, shmoo formation, and shmoo orientation. An attractive model is that Cdc24p acts as a selector switch that responds to input signals from bud site selection and mating projection orientation . We envision that the localization and activation of Cdc24p is essential for its function in both bud site selection and mating projection orientation. During budding it is likely that local activation of the G-protein Bud1p marks the site for bud formation . The GTP bound form of Bud1p binds Cdc24p and this interaction may be required for Cdc24p localization to the bud site. Interactions of Cdc24p with the bud site selection machinery dictate the site of mating projection growth in the absence of local activation of Cdc24p by Gβγ, such as in the case of far1 or cdc24 mutations or in the presence of saturating mating pheromone , wherein the mating projection forms adjacent to the previous bud. We show Bud1p becomes essential for shmoo formation specifically in the absence of signaling from Gβγ to Cdc24p. This demonstrates that the bud site selection machinery can function in shmoo formation. It is surprising that under these conditions, BUD1 functions in shmoo formation, while during budding it appears only to function in bud site selection and not bud formation. Interestingly, a specific role for BUD2 in bud formation has been observed in triple mutant combinations with Δcln1 and Δcln2 . A possible explanation for these different functions of BUD1 is that mating projection orientation is a continuous process, in contrast to bud site selection in which once a site for growth is chosen, subsequent directed growth is fixed to this site and there may no longer be a requirement for BUD genes. We attribute the role of BUD1 in shmoo formation to a synthetic effect with cdc24 / far1 suggesting this function of BUD1 is normally redundant yet revealed in the absence of Gβγ-mediated chemotropism. Recently it has been proposed that BUD1 is involved in cell fusion , yet the effects of Δbud1 we observe in cdc24-m1 mutants, i.e., the inability to form a shmoo, are unlikely to be a result of its role in fusion as we observe this morphological defect in response to mating pheromone without a mating partner. Furthermore, in contrast to the results of Elia and Marsh but in agreement with previous studies , Δbud1 does not result in a mating defect in our strain background. In addition, Δbud1 does not affect mating in the presence of saturating uniform mating pheromone concentration . Therefore while both mating in the presence of saturating pheromone or mating in a cdc24 or far1 mutant block chemotropic growth, at the molecular level these two situations are not equivalent and this difference is consistent with the suggestion that Cdc24p must be localized or locally activated to function properly . We imagine that during mating in saturating uniform pheromone concentrations, the Cdc24p-Far1p-Gβγ linkage is intact, but the external spatial signal is absent. In contrast, in a cdc24-m1 or Δfar1 mutant while the external signal is present, signaling from Gβγ to Cdc24p is prevented. Furthermore, the early localization of Cdc24p during shmoo and bud formation supports the proposed role of Cdc24p in linking a spatial landmark to polarity establishment. A simple mechanism for growth site selection during mating and budding is that a threshold level of locally activated Cdc24p is necessary to catalyze the GDP-GTP exchange of Cdc42p. This activation of Cdc24p is presumably generated in part by Bud1p during budding and switched to the region of the cell adjacent to the pheromone source by released Gβγ during mating. In such a mechanism, it would not be necessary to inhibit or erase the incipient bud site during mating as previously suggested . It is, however, possible that the binding of Cdc24p to Far1p results not only in an increased level of interaction with Gβγ but also a decrease in the amount of Cdc24p at the bud site, perhaps by decreasing its affinity for Bud1-GTP. We favor the notion of a balance between Cdc24p activation at the new bud site and at the region of the plasma membrane adjacent to pheromone source. We propose that Far1p serves to bias this equilibrium, i.e., shift the balance, towards the site for shmoo formation. Cells from a variety of organisms undergo polarized growth in response to external signals. For example, in C . elegans embryonic development it is the sperm entry site that determines antero-posterior axis . In Dictyostelium , cell aggregation occurs via cAMP-mediated chemotaxis and local activation of G-protein signaling events occurs in the absence of cell movement . Chemotaxis is necessary for cell migration responses for example of lymphocytes . Chemotropism is also essential for axonal guidance and neuronal growth cone remodeling and extension . Such processes are crucial for tissue and organ development. Many of these chemotactic and chemotropic processes appear similar to chemotropism during yeast mating, in that they depend on chemoattractant gradients that are recognized and transmitted by a molecular machinery including G-protein coupled receptors, rho-family GTPases, and their exchange factors. Chemotropic growth in yeast is therefore a suitable model for understanding the molecular basis of many different chemotropic and chemotactic processes. | Other | biomedical | en | 0.999997 |
10087264 | All plasmids used in this work are listed in Table I . Residues 503–968 of Sla2p were fused to the Gal4p DNA-binding domain in pAS1-CYH2 generating pDD373 (Yang, S., M.J.T.V. Cope, and D.G. Drubin, manuscript submitted for publication). This construct expresses a product of the expected size (71 kD), which is detected by anti-Sla2p antibody (data not shown). A yeast Y190 (Table II ) strain containing pDD373 was transformed with a library containing random cDNA fragments fused to the activating domain of Gal4p and was selected on synthetic medium lacking tryptophan, leucine, and histidine, and containing 50 μM 3-amino-1,2,4-triazole. Healthy colonies that also displayed β-galactosidase activity by filter-lift assay were selected and the activation-domain fusion plasmids were isolated and sequenced from the primer “HA_internal” (GCTTACCCATACGATGTT). Sequence alignments were performed using the ClustalW software package , implemented at the European Bioinformatics Institute web site at http://croma.ebi.ac.uk/clustalw/ . The phylogenetic tree was determined from the alignment data, again using ClustalW. An allowance was made for multiple substitutions . Information from intervals in the alignment for which gaps are found in some sequences was not included, in order to avoid the inappropriate weighting of some sequences. The tree was tested (1,000 trials) for branching order confidence by bootstrapping . Further information on this procedure (as applied to myosin motor domains) can be found on the World Wide Web at http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html . The ARK1 open reading frame (ORF) 1 was precisely replaced by a DNA fragment containing the HIS3 gene. The primers SY40 (GAGAAAGAAATATTACTCTGCATAATTAGGTATTTTAAGCAACCAGATAAATCAACCTGTGCGGTATTTCACACCGC) and SY41 (CATGTTACCAGCCTCTTCAGAGATCGATCCGGTTCTGTTGAGCCAAATACTCAGATTGTACTGAGAGTGCACC) were used to amplify a HIS3- containing DNA fragment from pRS313 using PCR. DDY426 was transformed with this fragment and the resulting transformants were screened for His + colonies. Replacement of the ARK1 ORF with the HIS3 -containing fragment was verified by PCR using the primers SY51 (CGGAGCTCGGCAACCTTCATGCCTTATG) and SY52 (CGTCTAGAGGAGAGCACAATCCAGC). The heterozygous ( ARK1/ark1 Δ::HIS3) diploid was sporulated and ark1 Δ::HIS3 MATa and ark1 Δ:: HIS3 MATα haploid cells were isolated. The PRK1 ORF was precisely replaced by a DNA fragment containing either the URA3 ORF or the LEU2 ORF. Primers SY44 (GTTGATCAAGATTATTTGTAACCTCCTATCTTTAGTTGAACTGATCCAAAAACACTGTGCGGTATTTCACACCGC) and SY45 (CATTTTGTATGACTTTTAATATTACATAGTCTATTATGTGTGAGAGCAAGTTTTAGATTGTACTGAGAGTGCACC) were used to PCR amplify URA3 -containing fragments from pRS316 or LEU2 -containing fragments from pRS315. Ura + or Leu + transformants of DDY426 were isolated and verified for replacement of the PRK1 gene by PCR using the primers SY61 (TGATGTGATAGTGGCACCAAAC) and SY62 (CGTATGCAGAGCGAAGGTCTT), or the primers SY45 and SY62. Diploids heterozygous at the PRK1 locus were sporulated and prk1 Δ::URA3 MATa , prk1 Δ::URA3 MATα , prk1 Δ::LEU2 MATa , and prk1 Δ::LEU2 MATα haploid were isolated. Finally, yeast in which the ARK1 ORF deletion mutation was marked with LEU2 was obtained by using the marker-swap method . The plasmid HL3 (a kind gift from F. Cross) was digested with ApaI and PstI to release the LEU2 -disrupted HIS3 gene. The digested DNA was used to transform DDY1407. Leu + /His − yeast were identified and the new LEU2 -marked disruption of the ARK1 gene was verified by PCR using the primers SY63 and SY51. A genomic fragment containing the ARK1 ORF, plus 453 upstream base pair and 250 downstream base pair, was amplified from the S . cerevisiae cosmid 70944 by high-fidelity PCR using the primers SY51 and SY52. This fragment was inserted between the XbaI and SacI sites of pRS315, creating the plasmid pDD382. This plasmid fully complemented the temperature-sensitive growth defects of ark1 Δ prk1 Δ double-null yeast. A genomic fragment containing the PRK1 ORF, plus 385 upstream base pair and 284 downstream base pair, was amplified from the S . cerevisiae cosmid 70775 between primers JC_YIL095w_4 (TAGAGCTCGTACTGATAGAGATTTCCG) and JC_YIL095w_5 (CATGTAGTCGACCCACAACGAAGCTGCCCAAG). The PCR product was digested and inserted between the SacI and SalI sites of pRS316, creating pDD556. This plasmid fully complemented the temperature-sensitive growth defects of ark1 Δ prk1 Δ cells. A 6-myc epitope-tag flanked by XmaI sites (from pDD557) was inserted into the XmaI site present 11 codons into the PRK1 ORF in pDD556, forming pDD558. This plasmid fully complemented the temperature-sensitive growth defects of ark1 Δ prk1 Δ double-null mutant cells. For rhodamine-phalloidin staining of filamentous actin when preservation of green fluorescent protein (GFP) fluorescence was not required, cells were grown to log-phase in YPD . To 1.5 ml of cells in YPD, 200 μl of a 37% formaldehyde solution was added and the culture was incubated at room temperature for 30 min to 1 h. The cells were washed two times in PBS containing 1 mg/ml BSA (PBS-BSA) and then resuspended in 50 μl PBS-BSA, to which 10 μl of rhodamine-phalloidin (Molecular Probes) solution was added (300 U in 1.5 ml methanol). After a 30-min incubation, the cells were washed three times in PBS-BSA and resuspended in mounting medium before visualization. When preservation of GFP fluorescence was required, cells were fixed in 2% formaldehyde for no longer than 30 min. For actin immunofluorescence, a 1:2,000 dilution of guinea pig anti– yeast actin serum was used . The myc epitope-tag was detected using a 1:50 dilution of rabbit polyclonal anti-myc antibodies ( Santa Cruz Biotech .). Sla2p, Sac6p, and cofilin were detected using affinity-purified antibodies raised in rabbit, at dilutions of 1:50, 1:100, and 1:200, respectively . Secondary antibodies were: 1:2,000 dilution of FITC-conjugated goat anti–guinea pig antibody (Cappel/Organon Technika Inc.), and 1:2,000 dilution of Cy3-conjugated goat anti–rabbit antibody ( Sigma Chemical Co. ). Fixation and permeabilization of yeast cells was performed as described by Ayscough and Drubin . Conventional light microscopy of fixed and fluorescently labeled cells was performed using a Zeiss Axioskop fluorescence microscope equipped with a Zeiss 100×/1.3 Plan-Neofluar oil-immersion objective and a Sony CCD camera controlled by Phase-3 software (Phase-3 Imaging Systems). Microscopy of living cells expressing GFP fusion proteins was performed using a Nikon TE300 ( Nikon ) equipped with a 100× Plan-Apo/1.4 objective and an Orca-100 cooled-CCD camera (Hamamatsu) controlled by Phase-3 software. Confocal light microscopy of rhodamine-phalloidin–stained yeast was performed using a Zeiss 510 laser-scanning confocal microscope. The ORFs for YNL020c ( ARK1 ) and YIL095w ( PRK1 ) were cloned into GFP expression vectors under control of the GAL1,10 promoter as follows. ARK1 was amplified by PCR using the primers JC_YNL020c_1 (GCTCTAGACTTATCCAAGGATAACTTTCG) and SY50 (CGTCTAGAATGAATCAACCTCAAATTGG) with pDD382 as template. The product was cloned into the XbaI site of pTS395, creating a plasmid (pDD555) encoding a chimeric protein with GFP at the COOH terminus of Ark1p. . PRK1 was amplified by PCR using the primers JC_YIL095w_1 (CGGGGATCCATGAATACTCCACAGATTAG) and JC_YIL095W_2 (GCTCTAGATTAAACTTTGCTGGGAAACC) with pDD556 as the template. The product was inserted between the BamHI and XbaI sites of pTS408 to create a plasmid (pDD554) encoding a chimeric protein with GFP at the NH 2 terminus of Prk1p. All PCR reactions were performed with low numbers of cycles and with accurate DNA polymerases (Pfu from Stratagene or Vent from New England Biolabs ). The Ark1p-GFP (pDD555) construct complemented the growth defects of ark1 Δ prk1 Δ double-mutant cells at both 15 and 37°C. The GFP-Prk1p construct (pDD554) was itself deleterious to growth when overexpressed, but slow-growing colonies could form when this fusion protein was overexpressed in ark1 Δ prk1 Δ double-null cells at 37°C, whereas a control plasmid lacking an insert does not allow growth at 37°C. An Abp1p-GFP fusion construct, a kind gift from Tim Doyle (Stanford University), was also used in these studies . Induction of GFP-tagged proteins from the GAL1,10 promoter was accomplished by first picking from solid media a single, isolated colony (containing the requisite vector) and inoculating it into liquid synthetic medium (SM) lacking uracil and containing 2% glucose. After growth overnight, a small sample was removed, washed in SM, and transferred to SM lacking uracil, but containing this time 2% raffinose and 2% galactose as carbon sources. After 8–10 h, the glucose repression is overcome and the GFP-tagged fusion constructs begin to be expressed. Cells were visualized 12–16 h after transfer to the raffinose-galactose–containing medium. The codon encoding lysine 56 of Ark1p in pDD382 was converted to a codon encoding an alanine (K56A), using the primer JC_Ark_ded (GTTGCATGCTTGGCCAGAGTCATTGTTC), in conjunction with the primer JC_Afl2_mut (GTACGCCAACTTGAGACCATGTAAC) with the “Transformer” site-directed mutagenesis kit ( Clontech ). In addition to making the K56A mutation, JC_Ark_ded introduces a unique MscI site. JC_Afl2_mut creates a silent mutation within the LEU2 ORF that eliminates a unique AflII site. These mutations are incorporated in the plasmid pDD559. The kinase-dead ARK1 was amplified as described above using SY50 and JC_YNL020c_1 and inserted into the XbaI site of pTS395 for expression as a GFP fusion under the control of the GAL1,10 promoter, creating pDD562. This plasmid was sequenced across the XbaI site at the 5′ end of ARK1 and the K56A mutation verified. The codon encoding lysine 56 of Prk1p in pDD556 was converted to a codon encoding an alanine (K56A) using the primer JC_Prk_ded (CATAGCATGCTTGGCCAGAGTCATTGTC), in conjunction with the primer JC_NcoI_mut (CTTGACTGATTTTTCAATGGAGGGCACAGTTAAG), again with the “Transformer” site-directed mutagenesis kit. In addition to making the K56A mutation, JC_Prk_ded introduces a unique MscI site. JC_NcoI_mut creates a silent mutation within the URA3 ORF that eliminates a unique NcoI site. These mutations are incorporated in the plasmid pDD560. The kinase-dead PRK1 was amplified as described above using JC_YIL095w_1 and JC_YIL095w_2 and inserted into the XbaI site of pTS408 for expression as a GFP fusion under the control of the GAL1,10 promoter, creating pDD561. This plasmid was sequenced across the BamHI site at the 5′ end of PRK1 and the K56A mutation verified. The plasmids pDD561 and pDD562 were transformed into DDY130 and expression of the kinase-dead variants of Ark1p and Prk1p were induced as described above. The cells were examined by fluorescence microscopy to verify Ark1p and Prk1p expression and localization, by immunofluorescence microscopy for actin distribution, and by differential interference-contrast (DIC) microscopy for cell appearance and morphology. Cells were tested for viability using the vital dye FUN-1 (Molecular Probes). The cells were grown to log-phase in minimal medium and FUN-1 was added to a final concentration of 1 μM. After rotation at 25°C for 30 min, the cells were washed once and then resuspended in 250 μl of minimal media for visualization. Living cells were able to internalize the stain to the vacuole, where it could be metabolized to form aggregates that fluoresced brightly when observed using a rhodamine filter set. Dead cells, by contrast, displayed a uniform distribution of dye throughout the cell and did not develop bright vacuolar aggregates. Sla2p/End4p/Mop2p is a component of the yeast cortical actin cytoskeleton implicated in the control of actin organization, endocytosis, and the maintenance of an ATPase at the plasma membrane . To identify proteins that interact with Sla2p and thus might be involved in the assembly or function of cortical actin patches, a fusion of residues 503–968 of Sla2p to the DNA binding domain of Gal4p was used to screen a two-hybrid library of cDNA fragments fused to the activation domain of Gal4p . Of the six clones identified, two were derived from the yeast gene YNL020c. One clone encoded residues 380–638, and the other encoded residues 218–552 of the protein. The remaining clones were single isolates and have not yet been pursued further. YNLO20c encodes a protein of 638 amino acids with a 300-residue region at the NH 2 terminus containing many elements conserved in serine-threonine kinases . Because of the findings presented below, we have named this gene ARK1 , for actin regulating kinase 1. Examination of the S . cerevisiae genome indicated that the putative protein kinase domain of Ark1p is very similar in amino acid sequence to the kinase domain of a protein encoded by the ORF YIL095w. This protein was found in a genetic screen for modifiers of mammalian p53 activity in yeast and has been named p53-regulating kinase 1, or Prk1p . The kinase domains of the two proteins are 73% identical at the amino acid level. Furthermore, a third S . cerevisiae protein, encoded by the unstudied ORF YBR059c, a Schizosaccharomyces pombe protein, and proteins from Caenorhabditis elegans , Arabidopsis thaliana , rat, and human were also found to be similar to Ark1p and Prk1p within the kinase domains, having between 30 and 48% identity to the Ark1p kinase domain. This level of identity is significantly higher than that observed with any other kinases retrieved from the databases, according to the BLAST algorithm . The rat and human proteins have been identified as cyclin-G–associated kinases (GAKs) and the human GAK has been shown to be a functional kinase in vitro . The kinase domains of these proteins were reported previously to be most similar to those of Nek1 and CDK2 . However, our analysis now places them in a separate family. We propose to call this the Ark family, after Ark1p, the first member of this family to be named for a biological process. A sequence alignment shows the similarities between the kinase domains of Ark1p, Prk1p, and six other Ark family members . The residues that are absolutely conserved in all serine-threonine kinases are present, but there are significant variations from the norm that are distinguishing features of the eight kinase domains shown here . Most notably, the largely invariant p-loop sequence (involved in binding the nontransferable phosphates of ATP) in other kinases conforms to the consensus GxGxxGxV, while the Ark family members have their own consensus S/EGGFA/SxVY (where x is any amino acid, S/E is serine or glutamate, and A/S is alanine or serine). There is a GxGxxG motif very near the NH 2 terminus of the GAKs which has led to the suggestion that these residues might constitute part of a p-loop . However, based on the alignment shown in Fig. 1 B, we feel that these residues are unlikely to comprise the true phosphate anchoring loop. This is because the first two glycines are separated by a proline, an amino acid never seen at this position in other kinases, and because other conserved features are present surrounding the S/EGGFA/SxVY motif, but not surrounding the GxGxxG motif in these kinases. A crystal structure will be required to be certain about this issue. Fig. 1 B also shows that several other residues are absolutely conserved in the kinase domains of the Ark family members but are rarely found in kinases not in this group. To demonstrate objectively that these eight putative kinases are more closely related to each other than to other serine-threonine kinases, we performed a further multiple alignment. This time, we also included representative kinases from each of the classes defined in Hardie and Hanks (alignment not shown). The resulting phylogenetic tree indicates that the eight kinase domains shown in Fig. 1 B constitute a new family within the superfamily of serine-threonine kinases . Although the kinase domains of Ark1p and Prk1p are highly similar, their nonkinase COOH-terminal domains lack detectable similarity along most of their lengths. Ark1p encodes a protein of 638 amino acids with a nonkinase COOH-terminal domain of ∼340 amino acids, while Prk1p encodes a protein of 810 amino acids with a nonkinase 510–amino acid COOH-terminal domain with no significant extensive similarity to that of Ark1p. However, a short conserved motif does exist close to the COOH terminus of both proteins. Ark1p contains two copies, and Prk1p contains one copy, of the conserved motif PxPPPKP. Proline-rich regions are known to mediate protein– protein interactions and can be bound by, for example, Src-homology 3 (SH3) domains. Several interactions between proline-rich motifs and SH3 domains are found among proteins found in yeast cortical actin patches, and it is possible that Ark1p and Prk1p also participate in such associations . To determine whether deletion of either the ARK1 or the PRK1 gene has a detectable effect on the actin cytoskeleton or on the growth characteristics of the cell, we replaced each of these genes individually with auxotrophic markers. Deletion of these genes singly caused little or no detectable defects in the actin cytoskeleton . However, growth rates were slightly lower than in the parental wild-type strain (1.8 h doubling time at 30°C for each single deletion mutant compared with 1.5 h for the wild-type [DDY130]). Whereas the effects of genetically removing either Ark1p or Prk1p from the cell were negligible , the effects of the removal of both proteins were profound . ark1 Δ prk1 Δ double mutants were inviable at 15 and 37°C, and grew optimally at 30°C. At 30°C, many of the double-mutant cells were substantially larger than the parental cells and had thicker bud necks . Moreover, the double-mutant cells displayed a severely abnormal actin cytoskeleton. Most cells in the population (85%) contained one or more large clumps of actin . Similar clumps are not seen in wild-type cells. These clumps contain filamentous actin because they stain brightly with rhodamine-phalloidin. Actin cortical patches and actin cables were still present, although the cortical patches no longer appeared uniform in size and they were not polarized. In addition, fewer cables could be observed in the double-mutant cells than in wild-type cells (50% of mutant cells contained visible cables versus 75% in the wild-type cells). Despite the severity of the actin defects, these cells did manage to form buds and divide. The doubling time of ark1 Δ prk1 Δ haploid cells at 30°C was ∼3 h, however, compared with a doubling time of 90 min for the parental wild-type cells. An increase over wild-type in the number of multinucleate cells was not observed. Shifting cells from 30 to 37°C or to 15°C did not change actin distribution noticeably. The phenotypes observed in the ark1 Δ prk1 Δ double-mutant cells were rescued when they were transformed with low-copy (CEN) plasmids bearing genomic fragments containing either ARK1 (pDD382) or PRK1 (pDD556) (data not shown). We then mutated in Ark1p and Prk1p a conserved lysine within the kinase domain that had been shown in a number of previous studies on other members of the protein kinase superfamily to result in a loss of protein kinase activity . For both Ark1p and Prk1p, a conserved lysine (K56) was changed to an alanine. When ark1 Δ prk1 Δ double-mutant cells were transformed with low-copy plasmids bearing the kinase-dead mutants of ARK1 (pDD559) and PRK1 (pDD560), the phenotypes described above were not rescued (data not shown). These data suggest that Ark1p and Prk1p are active kinases, and that their kinase activities are necessary for their in vivo function(s). To determine whether the actin clumps observed by rhodamine-phalloidin staining in ark1 Δ prk1 Δ cells were merely abnormal aggregates of filamentous actin derived from cables, or whether they also contained proteins normally found in cortical actin patches, we performed indirect immunofluorescence on these cells using antibodies directed against Sac6p (yeast fimbrin), Sla2p, and cofilin. All of these proteins are normally found in actin patches, Sac6p also associates with actin cables. All three of these proteins associated with the actin clumps in ark1 Δ prk1 Δ cells . Abp1p-GFP also localizes to these clumps in vivo (data not shown). In addition, all four proteins are found to be present in the cortical actin patches that remain in the ark1 Δ prk1 Δ cells. As has been observed in wild-type cells, the localization of cortical Sla2p patches was not always coincident with that of cortical actin patches (Yang, S., M.J.T.V. Cope, and D.G. Drubin, manuscript submitted for publication). The presence of cofilin, Sla2p, Sac6p, and Abp1p in the actin clumps of ark1 Δ prk1 Δ double-mutant cells is a characteristic shared with actin cortical patches. To determine whether these clumps were also associated with the cell cortex, we used confocal microscopy. The actin clumps were found associated with the cell cortex in only a minority of cases . Immuno-EM using anti-actin antibodies has verified this observation (data not shown). Furthermore, the confocal microscopy revealed that, while variable in size, the actin clumps could occupy a volume of the cytoplasm comparable to that of the nucleus. Thus, the clumps are not merely comprised of many actin patches aggregated at the cell surface. Rather, they are large masses of actin and other cortical patch proteins that accumulate in the absence of Ark1p and Prk1p. Since Ark1p was found as a protein that interacts with Sla2p, and since Ark1p and Prk1p were shown to affect profoundly the organization of cortical actin cytoskeleton proteins, we determined the subcellular localization of Ark1p and Prk1p. First, we placed a 6-myc epitope-tag close to the NH 2 terminus of each protein. In the case of Prk1p, this construct was capable of fully complementing the actin and growth phenotypes of the ark1 Δ prk1 Δ double-mutant cells when expressed from a low-copy plasmid under its own promoter. Indirect immunofluorescence of actin and of the myc epitope in cells expressing only the tagged version of Prk1p shows that this protein localizes to cortical actin patches . These patches were appropriately organized according to the cell-cycle stage, indicating that expression of the myc-tagged protein was not causing the formation of abnormal structures or interfering with actin localization. Epitope-tagged constructs of Ark1p appeared to be unstable. As an alternative approach to localizing Ark1p and to examine the in vivo localization of both Ark1p and Prk1p, we placed ARK1 and PRK1 in vectors that would express them as GFP fusion proteins under the control of the strong, inducible GAL1,10 promoter. When expressed in the presence of galactose, Ark1p-GFP was visible in cortical patch structures very similar in appearance and behavior (i.e., motility) to actin patches. When GFP and actin were both localized by indirect immunofluorescence, they were found to be coincident in patches at the cell cortex . Kinase-dead Ark1p-GFP, which does not perturb the actin cytoskeleton (see below), also localized to cortical actin patches. Thus, we conclude that Ark1p localizes to cortical actin patches. GFP-Prk1p was also found to colocalize with cortical actin patches . Elevated levels of Ark1p and Prk1p lead to the formation of delocalized actin patches and actin bars . Actin bars are intracellular aggregates of actin monomers and are therefore not labeled by rhodamine-phalloidin, a compound that binds to filamentous but not monomeric actin. Actin bars are in this respect different from the actin clumps observed in ark1 Δ prk1 Δ cells. Continued overexpression of either Ark1p or Prk1p leads to a variety of further and more severe effects . In the case of Prk1p, this ultimately leads to inviability of the cell population. The initial consequences of overexpression of either Ark1p or Prk1p included cells with abnormally shaped buds, and apparent septation defects, multiple buds, and/or severely abnormal internal structures. Cells with abnormal internal structures were determined to be dead using the vital dye FUN-1 . 4–6 h after induction of Prk1p-GFP overexpression, ∼35% of budded cells had abnormal buds and/or multiple buds, and ∼25% of cells were dead. Most dead cells contained actin bars. Continued growth of Prk1p-GFP–expressing cells (24 h) in galactose-containing media resulted in lethality for the majority of cells, although some did survive and micro-colonies were formed after 5–7 d growth at 30°C (data not shown). While overexpression of Ark1p caused similar bud morphology, septation, and multibudded phenotypes after 4–6 h after derepression , fewer dead cells were seen (15–20%) and this percentage did not exceed 25% after continued overexpression (24 h). The fact that Prk1p overexpression is lethal to most cells was independently demonstrated in this laboratory in a screen for proteins that cause death upon overexpression (DUO). The DUO screen was performed on yeast cells transformed with a galactose-inducible genomic library to select for transformants that grew on glucose plates (expression inhibited) and that died on galactose plates (expression induced). The PRK1 gene was identified in a subsequent visual screen as a gene that caused lethality and abnormal cell morphology when present at elevated levels. The overexpression phenotypes caused by this untagged Prk1p were identical to those described above. As reported above, the kinase activities of Ark1p and of Prk1p are necessary for either protein to rescue the phenotypes of ark1 Δ prk1 Δ double-mutant yeast. To test whether the overexpression phenotypes were also dependent on kinase activity, we placed the kinase-dead variants of the proteins, also as GFP-containing chimeras, under the control of the GAL1,10 promoter. Overexpression of the kinase-dead variant of Prk1p did not cause inviability . However, the kinase-dead variant of Prk1p retained the ability to cause delocalization of actin patches (data not shown). As mentioned above, the kinase-dead variant of Ark1p localized to cortical patches, but when present at elevated levels no longer caused delocalization of actin patches , nor did it cause abnormally budded or multibudded cells, death, or any of the phenotypes that were exhibited by cells containing elevated levels of wild-type Ark1p. Furthermore, using the kinase-dead mutant, the colocalization of Ark1p with cortical actin patches (and not actin cables) was confirmed under conditions where the actin cytoskeleton itself is unperturbed. The COOH-terminal, nonkinase domain of Ark1p interacts with Sla2 in the two-hybrid assay and Sla2p partially colocalizes with actin in cortical patches (Yang, S., M.J.T.V. Cope, and D.G. Drubin, manuscript submitted for publication). Thus, it was of interest to determine whether Ark1p and Prk1p were capable of being localized to cortical patches in the absence of Sla2p. Interestingly, both Ark1p and Prk1p were still found in patches at the cortex in sla2 Δ cells . However, localization of both proteins to cortical patches was reduced dramatically in abp1 Δ cells. Ark1p and Prk1p localize to cortical patches in sac6 Δ, srv2 Δ, and in rvs167 Δ cells. Yeast containing sac6 Δ, srv2 Δ, or rvs167 Δ mutations have abnormal actin cortical patch morphologies, whereas abp1 Δ yeast do not . Overexpression of Ark1p and Prk1p results in further disruption of actin in each of these null mutant strains (even in abp1 Δ cells); however, it is clear that Ark1p and Prk1p remain colocalized with actin in all the mutants tested, except abp1 Δ cells. The synthetic lethality that results when ark1 Δ and prk1 Δ are combined suggests that these proteins function in parallel to regulate an essential process or processes. To gain deeper insight into how Ark1p and Prk1p might regulate the actin cytoskeleton, we constructed a number of double mutants containing a null allele of ARK1 or of PRK1 , together with a null allele of a gene implicated in cortical actin cytoskeleton function. The genes chosen for testing were ABP1 , SAC6 , SLA1 , SLA2 , SRV2 , RVS167 , CRN1 , and AIP1 . Abp1p is an actin-binding protein that localizes to actin patches . Sac6p, the yeast fimbrin homologue, bundles actin filaments and localizes to both actin patches and to actin cables . Sla1p is an SH3 domain containing protein found in cortical actin patches . Null alleles of the SLA1 gene are synthetic lethal with null alleles of the ABP1 and RVS167 genes . SLA2 encodes a cortical actin-binding protein and mutant alleles of this gene are synthetic lethal with ABP1 null alleles. Mutations in the SLA2 gene lead to actin and endocytosis defects . Srv2p, the yeast homologue of CAP, binds to adenylyl cyclase, Abp1p, and actin monomers . Rvs167p is necessary for a normal actin cytoskeleton morphology and for endocytosis . Yeast coronin (Crn1p) binds tightly to actin and localizes to actin patches . Aip1p interacts with actin and cofilin, and localizes to actin patches (Rodal, A.A., J. Tetrault, P. Lappalainen, D.G. Drubin, and D.C. Amberg, manuscript submitted for publication). Table III summarizes the effects of combining null mutations in the above genes with null mutations in the ARK1 and PRK1 genes. In addition to showing a negative synergism with prk1 Δ, ark1 Δ also shows a synthetic genetic interaction with sac6 Δ. This was the only additional genetic interaction detected involving ARK1 . By contrast, prk1 Δ, as well as showing negative synergism with ark1 Δ and with sac6 Δ, shows synthetic genetic interactions with sla2 Δ and with abp1 Δ. From two separate crosses between different sla2 Δ and prk1 Δ strains (32 tetrads were dissected), >50% of predicted prk1 Δ sla2 Δ double-mutant spores were viable. Surviving double mutants were extremely temperature and cold sensitive and could not reliably be streaked to single colonies. Thus, we consider the prk1 Δ sla2 Δ combination to be lethal. We have identified a putative serine-threonine kinase that, in the yeast two-hybrid system, binds to the evolutionarily conserved, cortical actin-associated protein, Sla2p. We have subsequently assigned the gene name ARK1 (for actin-regulating kinase 1) to this locus. Ark1p and another S . cerevisiae protein, encoded by the PRK1 gene, have at their NH 2 termini predicted serine-threonine kinase domains of ∼300 amino acids that are >70% identical. Searches of sequence databases identified six other kinases that are highly similar, within their kinase domains, to Ark1p and Prk1p, but are dissimilar outside the kinase domains. Together, these eight proteins define a new family of serine-threonine kinases, which we have termed the Ark family. Two mammalian members of the Ark family were identified as cyclin-G–associated proteins , although there are no proteins with significant homology to cyclin-G in budding yeast. A third predicted serine-threonine kinase from S . cerevisiae , encoded by the ORF YBR059c, is a member of the Ark family of kinases and has been implicated in the yeast pheromone response pathway . The kinase domain of this protein is less similar to those of Ark1p and Prk1p than they are to each other. Since Ark1p and Prk1p are similar primarily only in their kinase domains, but both regulate the actin cytoskeleton organization in S . cerevisiae , it is also possible that Ark family kinases present in other organisms have a related role. Deletion of either the ARK1 or the PRK1 gene individually had no observed consequences with respect to actin distribution, cell morphology, or growth. By contrast, when deletions in the ARK1 and the PRK1 genes were combined, the effect was strongly deleterious. Yeast lacking both Ark1p and Prk1p were sensitive to high and low temperatures, were slow growing, and had severely disrupted actin cytoskeletons. It appeared that the majority of the filamentous actin in these cells was present in large clumps. These actin clumps also contain other proteins normally found in cortical actin patches, suggesting that the clumps result from a loss of actin patch regulation. The clumps are typically not cortical, although electron microscopy (not shown) suggests that they might maintain connection with the cortex. Therefore, the clumps might be formed by the inappropriate aggregation of many cortical patches/patch proteins at the cell cortex. Since cortical actin cytoskeleton proteins seem to be part of the endocytic machinery, actin patch aggregation at the cell cortex might in turn lead to invagination of the cell surface such that large clumps of cortical actin cytoskeleton proteins become inappropriately localized to the cytoplasm. Alternatively, the clumps might form as a result of detachment of patches from the cortex, or they might form as a result of inappropriate nucleation of patch assembly in the cytoplasm. The latter scenario would be possible if patch components are themselves recycled between an endocytic compartment and the plasma membrane, and loss of Ark1p and Prk1p function caused proteins responsible for nucleating patch assembly to be trapped in an endocytic compartment. The cellular defects caused by loss of both Ark1p and Prk1p are alleviated by expressing wild-type Ark1p or Prk1p, but not by expressing Ark1p or Prk1p carrying a mutation in a residue required for kinase activity. Overexpression of either wild-type kinase results in a number of actin and growth defects, but these defects are absent when kinase-dead Ark1p is overexpressed, and reduced when kinase-dead Prk1p is overexpressed. Thus, the function(s) of Ark1p and Prk1p appear to depend upon their ability to function as kinases. Ark1 and Prk1 fusion proteins localize to cortical actin patches. These two kinases are the first signaling proteins known to localize to actin patches. Because of their localization, they are candidates for downstream effectors of signaling pathways controlled by proteins such as Cdc42p that, although not localized in patches, appear to regulate the cortical actin cytoskeleton. Since Ark1p and Prk1p both need to be eliminated before significant defects in the actin cytoskeleton are observed, these kinases seem to have redundant functions. To gain deeper insights into the functional relationships between these kinases, we made a series of double mutants between the ark1 Δ or prk1 Δ mutants and mutants of genes encoding actin cortical cytoskeleton proteins. Any synthetic effects involving the ARK1 or the PRK1 genes are likely to be significant since ark1 Δ and prk1 Δ single-mutant cells are very similar to wild-type cells in terms of appearance and growth. Null mutants in ARK1 or in PRK1 both show synthetic defects in combination with null mutants in the SAC6 gene, encoding yeast fimbrin, an actin filament bundling protein . No other synthetic interactions involving ARK1 were observed. By contrast, prk1 Δ showed severe negative synergy in combination with sla2 Δ and with abp1 Δ. prk1 Δ and sla2 Δ were synthetic lethal. prk1 Δ abp1 Δ double-mutant cells were large and contained actin clumps very similar to those found in ark1 Δ prk1 Δ cells. abp1 Δ cells, like ark1 Δ and prk1 Δ single-mutant cells, are very healthy and normal in appearance. The pronounced synthetic phenotype of the prk1 Δ abp1 Δ double mutants thus reflects an extreme negative synergism. We conclude that Prk1p and Abp1p contribute to a critical process in a redundant manner, and that Abp1p may function with Ark1p because both proteins are redundant with Prk1p but not with each other. ark1 Δ sac6 Δ and prk1 Δ sac6 Δ cells were very large and temperature sensitive, but did not contain actin clumps (data not shown). The genetic interactions with sac6 Δ may reflect general additive effects of cytoskeleton mutants rather than specific functional relationships in patch regulation because the sac6 Δ mutation shows synthetic effects with a large number of mutant alleles of genes encoding components of the actin cytoskeleton , and because Sac6p is an actin filament bundling and stabilizing protein . A pronounced phenotypic synergy between mutant alleles of two genes can reflect at least two functional relationships between the products of the genes. First, it may indicate that they act in separate pathways that can each perform the same essential function. Second, it may indicate that the two gene products are components of an essential protein complex which can retain functionality in the absence of one, but not both, proteins. abp1 Δ, for example, is synthetic lethal with sla2 Δ. This could be interpreted to suggest that Sla2p and Abp1p function in separate pathways towards a common, essential process. However, there is evidence suggesting that Abp1p and Sla2p might function as part of the same protein complex. Abp1p interacts with Rvs167p and Srv2p via SH3 domain– poly-proline interactions , and Rvs167p interacts with Sla2p in a two-hybrid assay . Both proteins localize to cortical actin patches, and a specific domain in Sla2p performs an endocytosis function that is redundant with a function performed by Abp1p . Thus Abp1p, Sla2p, Rvs167p, and Srv2p have the potential to form a complex that may interact with and be regulated by Ark1p. Similarly, the genetic interactions involving Prk1p and Ark1p may reflect synergistic contributions to the integrity of a single protein complex, or, alternatively, parallel pathways . The lack of synthetic interactions between ark1 Δ and sla2 Δ, or between ark1 Δ and abp1 Δ, suggests formally that Ark1p, Sla2p, and Abp1p function in the same pathway. The fact that Ark1p and Sla2p interact in the two-hybrid assay supports this assessment. However, abp1 Δ and sla2 Δ are synthetically lethal, as mentioned above. Therefore, a more satisfactory interpretation of the current results would place Ark1p in a cortical complex together with Abp1p and Sla2p. In the absence of Sla2p, Ark1p is still capable of localizing to cortical patches, so Ark1p is obviously capable of interacting with other cortical patch components, possibly via its proline-rich motifs. Abp1p, on the other hand, is important for the localization of Ark1p to patches, so perhaps its SH3 domain is also capable of interacting with the proline-rich motif in Ark1p. prk1 Δ, in contrast to ark1 Δ, shows synthetic effects with both sla2 Δ and with abp1 Δ. Therefore, Prk1p may either function in a separate pathway that operates in parallel to the Ark1p pathway to regulate the cortical actin cytoskeleton, or it may impinge upon the same complex in a different manner. The fact that combining null mutations in the SLA2 and PRK1 genes leads to more deleterious effects than result from combining null mutations in the ARK1 and PRK1 , or in the ABP1 and PRK1 genes, suggests that Sla2p has additional functions to those that involve Ark1p and Abp1p. As mentioned above, placing Prk1p in a separate pathway from Ark1p, however, does not exclude the possibility that Prk1p interacts with a potential Abp1p/ Rvs167p/Srv2p/Sla2p complex. Prk1p localizes to cortical actin patches, and, as with Ark1p, it shows a dependency on Abp1p (but not Sla2p, Rvs167p, Sac6p, or Srv2p) for normal localization to these patches, implying that it too may associate with such a complex. However, since deletion of the ABP1 gene does not result in a phenocopy of the ark1 Δ prk1 Δ double deletion and does not eliminate the effects of Ark1p or Prk1p overexpression, it is reasonable to assume that localization at a reduced level allowed by interaction with, for example, Sla2p, is sufficient for Ark1p and Prk1p function. Alternatively, it might be that localization of these kinases to cortical patches is not required for Ark1p and Prk1p to fulfill their cellular functions. What might be the phosphorylation targets of Ark1p and Prk1p? Our evidence strongly suggests that both Ark1p and Prk1p are functional kinases in vivo (see above). Other Ark family kinases have also been shown to be functional protein kinases in vitro . One putative target is Sla2p, because it interacts with Ark1p in the two-hybrid system. Sla2p is a phosphoprotein in vivo, but phosphorylation of Sla2p is not eliminated in ark1 Δ prk1 Δ cells (data not shown). Results published while this manuscript was under revision indicate that Prk1p regulates by phosphorylation the activity of Pan1p . Pan1p is a yeast homologue of the mammalian Eps15 proteins that play an important role in endocytosis . Prk1p is also capable of phosphorylating the cortical actin patch protein, Sla1p . Pan1p is an essential protein, a temperature-sensitive mutation ( pan1-4 ) which causes the appearance of actin clumps similar to those observed in ark1 Δ prk1 Δ cells under nonpermissive conditions . The similar phenotypes of pan1-4 and ark1 Δ prk1 Δ cells suggest that Pan1p might be regulated by Ark1p and by Prk1p. Since actin clumps do not appear in ark1 Δ or prk1 Δ single-mutant cells, Pan1p might be phosphorylated by Ark1p in the absence of Prk1p and vice versa . While our studies, together with those of Zhang and Cai, suggest that both Ark1p and Prk1p play important roles in Pan1p regulation, our genetic analysis is most consistent with the possibility that Prk1p but not Ark1p regulates Sla1p. We base this conclusion on the observation that null mutations in the PRK1 gene, but not in the ARK1 gene, have similar genetic interactions as those exhibited by null alleles of the SLA1 gene. Ark1p and Prk1p clearly play a critical role in regulating actin distribution in vivo. This conclusion is based on several different criteria: effects of null mutations, localization, overexpression effects, and genetic interactions with other genes encoding proteins known to be involved in modulating actin distribution in yeast. Elucidation of the signaling pathways in which Ark1p and Prk1p are involved and the identification of upstream and downstream components of those pathways are now important goals. | Other | biomedical | en | 0.999998 |
10087265 | S . cerevisiae strains used in this study are listed in Table I . Yeast growth media, molecular biological techniques, and genetic manipulations were as described previously . Yeast transformation procedures were performed using the lithium acetate method . Where indicated, rich medium, consisting of yeast extract, peptone, and dextrose, was supplemented with benomyl ( DuPont ), dissolved in DMSO, to a final concentration of 10, 20, or 30 μg/ml. Sensitivity of wild-type yeast strains to these concentrations of benomyl varied between preparations of benomyl containing agar plates and, hence, growth comparisons were always performed on the same plate. Sensitivity also varies dramatically between growth temperatures (i.e., at 23°C strains are much more benomyl sensitive than at 30°C). A strain containing the VIK1::3XHA allele was constructed by the PCR-epitope tagging method described previously . The primers 5′-TATTAACGATTTCAGAAGAAGTTCAAACACAACTTTGTAAAAGAAAGAAAAAGCTCACTAGGGAACA-AAAAGCTGG-3′ and 5′-CTTATTTGTTTCATATCTAAATGGCTGTG TTAAGAAAGACGATAATG TGACCGAGC TTAC TATAGG-GCGAATTGG-3′ were used in a PCR reaction with pMPY-3XHA as the template. The resulting 1.5-kb PCR product contains the URA3 gene flanked by direct repeats encoding three copies of the hemagglutinin (HA) epitope and contains 59 bp of sequence from the 3′ end of the VIK1 gene at one end and 59 bp of sequence downstream of, and including, the VIK1 translation termination codon at the other end. This fragment was used to transform yeast strain Y1731, and transformants were selected on synthetic complete medium lacking uracil. Correct integration into the 3′ region of the VIK1 locus was confirmed by PCR analysis with primers to sequences flanking the site of insertion. Transformants with a correct 3XHA-URA3-3XHA integration were then incubated on plates containing 5-fluoroorotic acid to select for loss of the URA3 marker by recombination between the repeated 3XHA regions. The resulting VIK1::3XHA allele was confirmed by PCR and immunoblot analysis. This allele does not display any vik1Δ phenotypes; growth of VIK1::3XHA strains in the presence of benomyl is identical to wild-type strains, and the temperature-sensitive growth defect of cik1Δ mutants is the same in a VIK1 or VIK1:: 3XHA background. Cells were grown in rich liquid medium to mid-logarithmic phase (OD 600 = 0.5–0.8), and a total of 10 OD 600 units of cells were collected by centrifugation, washed, and resuspended in 100 μl lysis buffer (1 M NaCl, 10 mM EDTA, 2 mM EGTA, 5% glycerol, 40 mM Tris-HCl, pH 7.5, containing 1 μl yeast protease inhibitor cocktail ( Sigma Chemical Co. ) and 200 μM PMSF). When indicated, cells were first washed twice with fresh medium, resuspended in medium containing 5 μg/ml α-factor mating pheromone ( Sigma Chemical Co. ), and incubated for 2 h before harvesting. Cell lysates were prepared in Eppendorf tubes using zirconia/silica beads (Biospec Products) with 40-s pulses of vortexing separated by incubations on ice; this procedure was repeated 6–8 times. Lysates were then centrifuged for 10 min at 6,500 g , and a 10-μl aliquot was removed for immunoblot analysis. For immunoprecipitations, the remaining cell lysate and beads were washed with 500 μl lysis buffer containing detergents (1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) without NaCl for 20 min on a roller drum at 4°C. Lysates were then cleared of unlysed cells and cell debris by centrifugation for 10 min at 6,500 g and ∼500 μl of supernatant was transferred to a new tube. The 500 μl lysate was brought to a 1-ml vol with the same lysis buffer plus detergents (100 mM final NaCl concentration). The cell lysate was then precleared for 1 h by incubation with 20 μl of a 1:1 slurry of protein A/G–agarose (Pierce) and TBS (150 mM NaCl, 50 mM Tris-HCl, pH 8.0). Immunoprecipitations were performed by incubation of cell lysates with either 2 μl mouse monoclonal anti-HA antibodies (12CA5 from BABCO), 10 μl rabbit polyclonal anti-Cik1p antibodies , or 5 μl rabbit polyclonal anti-Kar3p antiserum for 2 h. 20 μl protein A/G–agarose was then added and incubated for 1 h before collection by centrifugation at 2,000 g for 1 min. The protein A/G–agarose antibody complexes were then washed twice with 1 ml TBS containing detergents and protease inhibitors (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 μl Sigma yeast protease inhibitor cocktail, 200 mM PMSF) and once with 1 ml TBS plus protease inhibitors. The final pellet was resuspended in 30 μl Laemmli sample buffer . Proteins from cell lysates and immunoprecipitations were denatured by incubation at 90°C for 10 min before electrophoretic separation in either 8% or 10% SDS–polyacrylamide gels. For attempts to detect Vik1p-3XHA in mating-pheromone treated cells, a threefold larger volume of cell lysate was loaded than for vegetative cell lysates (data not shown). Proteins were then transferred to Immobilon-P membranes ( Millipore ) for immunoblot analysis with either mouse monoclonal anti-HA antibodies (12CA5 from BABCO), rabbit polyclonal anti-Cik1p antibodies , or a crude IgG fraction of the rabbit polyclonal anti-Kar3p antiserum . Reactive protein bands were then detected with alkaline phosphatase-conjugated secondary antibodies ( Amersham ) and the CDP Star detection reagent ( Boehringer Mannheim ). Overexposure of all blots failed to detect Vik1p-3XHA in cell lysates from mating-pheromone treated cells. Indirect immunofluorescence was performed as described previously . Due to sensitivity of the HA-epitope to formaldehyde fixation, mid-logarithmic phase cells were fixed with 3.7% formaldehyde for only 15 min. They were then washed twice with solution A (1.2 M sorbitol, 50 mM potassium phosphate buffer, pH 6.8), and spheroplasts were prepared by incubating cells in solution A containing 5 μg/ml Zymolyase 100T, 0.03% glusulase, and 0.2% 2-mercaptoethanol at 37°C for 15 to 30 min. Spheroplasted cells were then washed and resuspended in solution A and placed onto poly( l -lysine)- coated slides. Vik1p-3XHA and Kar3p-HAT were detected by incubation overnight at 4°C with preabsorbed mouse monoclonal anti-HA primary antibodies (16B12 from BABCO) diluted in PBS/BSA. Bound mouse anti-HA antibodies were then detected by incubation for 90 min at room temperature with preabsorbed CY3-conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratory, Inc.). Microtubules were detected by incubation with rabbit anti-yeast β-tubulin (Tub2p) primary antibodies followed by incubation with FITC-conjugated goat anti–rabbit secondary antibodies. After both primary and secondary antibody incubations, slides were washed twice with PBS/BSA and twice with PBS/BSA plus 0.1% NP-40. Finally, slides were mounted in 70% glycerol, 2% n -propyl gallate, and 0.25 μg/ml Hoechst 33258 to preserve the preparation and stain DNA for localization of nuclei. Photographs of representative cells stained with anti-HA antibodies, anti-Tub2p antibodies, and/or Hoechst 33258 were taken, and composite figures were produced and processed using Adobe Photoshop version 3.0 (Adobe Systems, Inc.). Processing procedures were identical for each photo of a particular staining method within a composite figure. A strain containing the KAR3::HAT allele was constructed using a transposon insertion technique described previously . The entire KAR3 coding region was cloned into the vector pHSS6 and subjected to transposon mutagenesis in Escherichia coli as described . Plasmid DNA was prepared from selected strains and digested with NotI, producing a fragment containing the mTn-3xHA/ lacZ transposon inserted randomly into the KAR3 gene. These fragments were then used to transform yeast strain Y1869, using the URA3 marker encoded by the transposon for selection. Transformants with in-frame transposon insertions into the KAR3 genomic locus were selected by detection of β-galactosidase activity as described . Cre recombinase-mediated excision, leaving only the 93 amino acid HA/transposon tag (HAT) inserted, was induced by growth on galactose (pB227 contains cre under control of a galactose-inducible promoter). Recombinants were selected on 5-fluoroorotic acid. Strains with in-frame HAT insertions were then tested for kar3Δ mutant phenotypes, such as defects in karyogamy and temperature sensitivity. PCR and sequence analysis was performed on DNA from fully complementing strains in order to determine the site of insertion within the KAR3 gene. Strain Y1870 contains a KAR3 allele with a HAT insertion after the codon for S 68 , and does not exhibit any kar3Δ phenotypes. Y1870 was then crossed with Y1864 to yield Y1871. Y1871 was then sporulated and a MATα KAR3::HAT cik1Δ::LEU2 segregant was isolated. The VIK1 gene was disrupted with the HIS3 marker in Y1870 yielding Y1752 (see below). Y1752 was then crossed with Y1751 yielding Y1753. Y1753 haploid segregants were used to analyze Kar3p-HAT localization in wild-type, cik1Δ , vik1Δ , and cik1Δ vik1Δ strains. Sequence of the VIK1 gene was acquired from the Saccharomyces Genome Database (ORF designation YPL253c; Stanford University). A 2,920-bp region, from the MscI site 5′ of the predicted VIK1 translation start site to the SpeI site 3′ of the VIK1 translation termination site, was PCR amplified from yeast genomic DNA and cloned into pBluescript SK (Stratagene), replacing its EcoRV-SpeI fragment (pSK- VIK1 ). The SalI-SacII fragment from this plasmid, containing the VIK1 gene and flanking sequences, was cloned into the SalI-SacII site of the CEN plasmid pRS316 . This plasmid was linearized by deleting the NruI-AflII fragment containing the entire VIK1 ORF, then gap repaired in a wild-type yeast strain . This CEN plasmid encoding wild-type VIK1 was used for subsequent phenotypic analysis. VIK1 disruption constructs were made by replacing the NruI-AflII fragment of pSK- VIK1 with the SmaI fragment from either pJA50 ( HIS3-Km r ) or pJA53 ( URA3-Km r ) . SalI-XbaI fragments from these plasmids, containing either the HIS3 or URA3 selectable markers flanked by the 5′ and 3′ noncoding regions of VIK1 , were used to transform yeast strains Y270, Y818, and Y1870 yielding vik1Δ strains Y1733, Y1748, and Y1752, respectively. CIK1 and KAR3 disruptions were made as previously described . The cik1-Δ3::LEU2 construct was used to transform strains Y817 and Y1744 yielding cik1Δ strains Y1758 and Y1745, respectively. The kar3-Δ4::URA3 construct was used to transform strains Y818 and Y1744 yielding kar3Δ strains Y1759 and Y1750, respectively. A database homology search with the Cik1p amino acid sequence identified an open reading frame (ORF) on chromosome XVI of the S . cerevisiae genome (BLAST search of predicted translation products of S . cerevisiae ORFs; Saccharomyces Genome Database ORF designation YPL253c) predicted to encode a protein with significant homology to Cik1p. This gene was named VIK1 , for vegetative interaction with Kar3p (see below). VIK1 is predicted to encode a protein of 647 amino acids, with an overall sequence identity of 24%, and similarity of 37%, to Cik1p. The Vik1p sequence also shares structural similarity to Cik1p. It contains a predicted α-helical coiled-coil domain (amino acids 80–385) of the same length and location as that found in Cik1p ; both proteins contain a short break of 40 amino acids in their coiled-coil regions. This break and an 80 residue NH 2 -terminal globular domain are the most divergent regions of the two proteins (16% identity). One notable difference in the amino terminal domain is that Vik1p lacks a recognizable nuclear localization signal found in Cik1p . Finally, the COOH-terminal globular domains of the two proteins share two regions of 134 and 43 amino acids in length with 25% identity each. Therefore, Vik1p shares sequence and structural homology to Cik1p which is not confined to their coiled-coil domains. To characterize the Vik1 protein by immunoblot and immunofluorescence analysis, DNA encoding a triple HA-epitope tag was integrated into the COOH-terminal coding region of the VIK1 genomic locus (see Materials and Methods). The resulting VIK1::3XHA fusion allele fully complements VIK1 function (see Materials and Methods). Immunoblot analysis using anti-HA monoclonal antibodies detects a protein of 92-kD in cell lysates and anti-HA immunoprecipitations from VIK1::3XHA strains . This molecular mass is close to the predicted 76-kD of Vik1p plus the triple-HA epitope tag. This fusion protein is not detected in cell lysates or anti-HA immunoprecipitations from VIK1 untagged strains . Therefore, the VIK1::3XHA allele is expressed during vegetative growth and produces a functional protein of the expected size. The Vik1p-3XHA protein is not detected in cell lysates or anti-HA immunoprecipitations from cultures first exposed to the α-factor mating pheromone . In contrast, Cik1p greatly increases in abundance upon exposure to α-factor . Consistent with this result, the region upstream of the VIK1 ORF does not contain any predicted pheromone-response elements . However, the pheromone-inducible CIK1 and KAR3 genes each contain multiple pheromone-response elements in their 5′ noncoding regions . Thus, since Vik1p is not present during mating-pheromone induced differentiation, it is not likely to have a significant role during mating. Cik1p physically associates with Kar3p , and Vik1p shares sequence and structural similarity to Cik1p; we therefore tested whether Vik1p could also interact with Kar3p. Cell lysates were prepared from wild-type, kar3Δ , and cik1Δ strains expressing either the VIK1:: 3XHA allele or untagged VIK1 . Proteins from these cell lysates were separated by SDS-PAGE or first immunoprecipitated with anti-Kar3p polyclonal antibodies . Immunoblot analysis using anti-HA antibodies detects Vik1p-3XHA in cell lysates from wild-type, kar3Δ , and cik1Δ strains containing the VIK1::3XHA allele but not in untagged strains . Therefore, Vik1p stability is not affected by the absence of either Kar3p or Cik1p. Anti-HA immunoblots of proteins immunoprecipitated with anti-Kar3p antibodies detects Vik1p-3XHA from a wild-type VIK1::3XHA strain but not from a kar3Δ VIK1:: 3XHA strain or an untagged strain . Therefore, immunoprecipitation data indicate that Kar3p and Vik1p physically interact. Furthermore, the Kar3p-Vik1p complex is quite stable, as cell lysates were prepared in a 1 M NaCl solution (see Materials and Methods). Vik1p-3XHA is also detected in anti-Kar3p immunoprecipitations from a cik1Δ VIK1::3XHA strain , indicating that Cik1p is not required for this interaction between Kar3p and Vik1p. Furthermore, an association between Vik1p and Cik1p is not detected by immunoprecipitation experiments. Vik1p-3XHA is not precipitated from VIK1::3XHA cell lysates by anti-Cik1p polyclonal antibodies , and, reciprocally, Cik1p is not precipitated from these cell lysates with anti-HA antibodies . Protein preparations from vegetatively growing cells or α-factor–treated cells produce the same results. Thus, Vik1p and Cik1p do not appear to be part of the same complex. Together, these results suggest that Vik1p and Cik1p interact with Kar3p separately, and, therefore, form two different complexes during vegetative growth. To determine the subcellular localization of Vik1p, asynchronous cultures of VIK1::3XHA and VIK1 untagged strains were fixed briefly with formaldehyde and analyzed by immunofluorescence using anti-HA and anti-tubulin antibodies (see Materials and Methods). VIK1::3XHA cells display anti-HA staining concentrated at the yeast microtubule-organizing centers, or spindle-pole bodies (SPBs), in all stages of the cell cycle . The position of the SPBs correspond to the brightest foci of anti-tubulin staining. Anti-HA staining at the SPBs is not detected in VIK1 untagged cells . Moreover, detection of Vik1p-3XHA at the SPBs does not require costaining with anti-tubulin antibodies; staining of dots at the edges of both preanaphase and anaphase nuclei is still observed in control cells not stained with anti-tubulin antibodies . Vik1p-3XHA staining is brightest in preanaphase cells with short spindles, where localization is clearly concentrated at the spindle poles . Cells in the G1 phase of the cell cycle, scored as unbudded cells with a single SPB, display SPB staining that is more faint and not observed in every cell (staining observed in 60% of G1 cells; n = 100). Finally, cells in early or late anaphase, as scored by elongated spindles and/or elongated nuclei penetrating the bud neck, display faint staining concentrated at the spindle poles . Anti-HA staining is not detected along the lengths of spindle or cytoplasmic microtubules in VIK1::3XHA cells. Short fixation times due to sensitivity of the HA-epitope to formaldehyde leads to a diminished number of cytoplasmic microtubules. However, in cells that contain cytoplasmic microtubules, only staining at the SPBs is observed; microtubule staining is not evident. These staining patterns are independent of ploidy, and the nature and position of the epitope tag. VIK1::3XHA/VIK1::3XHA homozygous diploids, and cells expressing a VIK1::3Xmyc NH 2 -terminal fusion allele stained with anti-myc monoclonal antibodies, exhibit identical patterns to those found in VIK1::3XHA haploid cells (data not shown). The localization of Vik1p at, or near, the SPBs at various levels throughout the vegetative cell cycle is very similar to that reported for fusion proteins of Kar3p with either β-galactosidase or HA . However, in pheromone-treated cells, when Kar3p localizes to the SPB and cytoplasmic microtubules , Vik1p-3XHA staining above background is not detected (data not shown). This is consistent with immunoblot data demonstrating the absence of Vik1p in cell lysates from α-factor–treated cells , and further suggests that Vik1p functions in vegetative cells, but not mating cells. Since Kar3p and Vik1p physically associate and have similar localization patterns, we investigated whether the localization of Vik1p to the SPBs was dependent on the presence of Kar3p. Therefore, Vik1p-3XHA localization was analyzed by anti-HA immunofluorescence in kar3Δ cells. Whereas Vik1p-3XHA concentrates at the SPBs in wild-type cells, it does not localize to the SPBs at any stage of the cell cycle in the kar3Δ mutant. Instead, the protein is present in cytoplasmic patches that appear to be excluded from the nucleus . This pattern is also observed with anti-myc staining in kar3Δ cells containing the VIK1::3Xmyc allele (data not shown). These cytoplasmic patches are not detected in kar3Δ VIK1 untagged strains . SPB localization of Vik1p-3XHA can be restored by introducing a CEN plasmid containing KAR3 into the kar3Δ VIK1:: 3XHA strain . This plasmid also restores normal spindle length, since disruption of KAR3 results in accumulation of cells with short spindles compared with wild-type cells . Therefore, the Kar3p-Vik1p association is required for localization of Vik1p to the SPB. Cik1p also localizes to the SPB in a Kar3p-dependent manner , and, like kar3Δ mutants, cik1Δ mutants accumulate cells with short mitotic spindles . Therefore, in order to determine if Cik1p was involved in localizing Vik1p to the SPB or if the short spindle phenotype was responsible for Vik1p mislocalization in kar3Δ mutants, we analyzed Vik1p-3XHA localization in a cik1Δ mutant. In contrast to kar3Δ cells, cik1Δ cells display Vik1p-3XHA staining concentrated at the SPBs during all stages of the cell cycle, as observed in wild-type cells. Furthermore, HA staining is brightest at the poles of the short preanaphase spindles present in cik1Δ cells , indicating that the short spindle phenotype alone is not the cause of mislocalization of Vik1p. Therefore, these immunolocalization data support the immunoprecipitation experiments; Vik1p does not appear to be part of the same complex as Cik1p. Furthermore, Vik1p localization to the SPBs does not require Cik1p function. Cik1p is required to localize Kar3p to the SPB and cytoplasmic microtubules during mating but is not required for Kar3p localization to the mitotic spindle poles . This suggests that in vegetative cells, Kar3p can either target to the SPBs by itself or interacts with another protein that is responsible for its concentration at the SPBs. Based on its localization and association with Kar3p, Vik1p could fulfill this function. Therefore, Kar3p was localized in the absence of Vik1p, Cik1p, and both Vik1p and Cik1p. The subcellular localization pattern of Kar3p has been determined previously using fusion proteins that do not fully complement all of the kar3Δ phenotypes . To create a fully complementing HA epitope-tagged version of Kar3p, we used a transposon insertion technique described previously . Using this technique we created several strains containing in-frame insertions of the HAT into random regions of the KAR3 genomic locus. We then tested these strains for several kar3Δ phenotypes, such as defects in karyogamy and meiosis, slow growth, temperature-sensitive growth, enhanced cytoplasmic microtubules, and the ability to properly localize Vik1p-3Xmyc to the SPBs . Fully complementing alleles were sequenced and found to lie within the region encoding the NH 2 -terminal globular domain of Kar3p (data not shown). A strain containing one of these KAR3:: HAT alleles (encoding Kar3p with the 93–amino acid HAT insertion at S 68 ) was used for immunofluorescence analysis with anti-HA antibodies. Localization of Kar3p-HAT was examined in wild-type, cik1Δ , vik1Δ , and cik1Δ vik1Δ strains. Asynchronous cultures of haploid segregants from a cik1Δ/CIK1 vik1Δ/ VIK1 KAR3::HAT/KAR3::HAT diploid strain were fixed and prepared for immunofluorescence with anti-HA and anti-tubulin antibodies (see Materials and Methods). KAR3:: HAT wild-type cells, in all stages of the cell cycle, display anti-HA staining concentrated at the SPB region, as well as faint patches confined to the nucleus (as determined by colocalization with anti-tubulin and Hoechst staining, respectively). These fluorescence patterns are not observed in KAR3 untagged strains, in which background staining is restricted to the cytoplasm . Consistent with previous Kar3p localization studies , Kar3p-HAT staining is brightest at the poles of preanaphase spindles . Most cells in G1 (80%, 100 G1 cells counted) and anaphase (86%, 100 anaphase cells counted) also display Kar3p-HAT localization at the SPBs, but this staining is fainter than that observed in preanaphase cells (data not shown). In contrast to previous studies, Kar3p-HAT localization along spindle microtubules is not detected in any stage of the cell cycle. When cytoplasmic microtubules are observed, in wild-type cells or the mutant cells described below, Kar3p-HAT localization is not detected along their lengths. Hence, Kar3p is most abundant at the SPBs of cells throughout the cell cycle. The effect of disrupting Vik1p and Cik1p function on Kar3p-HAT localization was determined. Since wild-type localization is most obvious in preanaphase cells, we quantified Kar3p-HAT staining patterns in cells from asynchronous cultures at this stage (Table II ). Similar to wild-type strains, KAR3::HAT cik1Δ cells display anti-HA staining at the SPBs during all stages of the cell cycle; again, the brightest staining is observed at the poles of the preanaphase spindle . However, the nuclear patch staining detected in wild-type cells is qualitatively diminished in the cik1Δ mutant. Therefore, as described previously , Cik1p is not required to localize Kar3p to the SPBs during the vegetative cell cycle. In contrast, Kar3p-HAT localization during vegetative growth is dramatically altered in vik1Δ cells . Kar3p-HAT is no longer concentrated at the SPBs in these cells; instead, it predominantly displays a bright nuclear patch localization in all vik1Δ cells. Moreover, staining along the lengths of spindle microtubules is detected in many cells . Some vik1Δ cells also display faint staining in the vicinity of the SPBs, but not along spindle microtubules (22% of preanaphase cells; Table II ). Therefore, although vik1Δ cells may retain some Kar3p at the SPBs, Vik1p is required to concentrate, or restrict, Kar3p localization to the SPBs. To determine if the spindle and faint SPB localization of Kar3p detected in vik1Δ cells is due to Cik1p function, we localized Kar3p-HAT in a cik1Δ vik1Δ double mutant. Kar3p-HAT localization is even more aberrant in the double mutant, with the nuclear patch staining distributed more diffusely throughout the nucleus in all cells . Detection of Kar3p-HAT along the spindle or at the SPBs in these cells is greatly reduced compared with the vik1Δ mutant (spindle and faint SPB localization is observed in 7 and 9%, respectively, of cik1Δ vik1Δ preanaphase cells; Table II ). Introduction of a CEN plasmid encoding VIK1 into either vik1Δ or cik1Δ vik1Δ mutants completely restores Kar3p-HAT concentration at the SPBs (data not shown). Therefore, during vegetative growth, Vik1p is primarily responsible for SPB targeting, or retention, of Kar3p. Cik1p may mediate Kar3p localization to less concentrated sites within the nucleus, such as the spindle and/or nuclear patches. Cik1p and Vik1p exhibit differences in their expression patterns, and deletion of CIK1 or VIK1 results in unique effects on Kar3p localization. Therefore, the phenotypes resulting from disruption of VIK1 and CIK1 were compared. Suprisingly, vik1Δ mutants grow similar to wild-type strains at all temperatures. Using a sectoring assay described previously , we determined that, unlike cik1Δ mutants that have a severe chromosome loss defect , vik1Δ mutants display only a threefold increase in frequency of chromosome loss per cell division relative to wild-type strains (data not shown). Furthermore, consistent with its absence in mating-pheromone treated cells, vik1Δ mutants are not defective in karyogamy, as determined by qualitative mating assays. However, compared with wild-type strains, vik1Δ mutants are resistant to the microtubule-depolymerizing drug benomyl . The benomyl resistance phenotype always segregates with the vik1Δ mutation (11 tetrads analyzed). To analyze whether CIK1 and VIK1 are functionally redundant, we tested whether expression of VIK1 from a high copy plasmid could suppress the temperature-sensitive growth defect of a cik1Δ mutant. cik1Δ strains containing either the VIK1 plasmid or vector alone are equally affected by growth at the restrictive temperature (data not shown). A cik1Δ mutant was then mated with a vik1Δ mutant, and the resulting heterozygous diploid strain was sporulated. Haploid segregants from tetratype tetrads were then analyzed . Wild-type, cik1Δ , vik1Δ , and cik1Δ vik1Δ segregants all display similar growth on rich medium at 23°C . However, cik1Δ mutants are temperature sensitive for growth at 37°C , whereas vik1Δ mutants grow like wild-type strains . Suprisingly, cik1Δ vik1Δ double mutants grow substantially better at 37°C than cik1Δ mutants . This result indicates that disruption of VIK1 partially suppresses the temperature-sensitive growth defect of cik1Δ mutants. The temperature-sensitive growth defect can be restored to a cik1Δ vik1Δ double mutant by introduction of a CEN plasmid encoding VIK1 (data not shown). Deletion of VIK1 also partially suppresses the mitotic delay of cik1Δ mutants, as scored by the percentage of large budded cells with a single preanaphase nucleus in logarithmic phase cultures growing at 30°C . 54% of cells from cik1Δ cultures exhibit the mitotic delay phenotype, whereas only 36% of cells from cik1Δ vik1Δ cultures have this phenotype. Wild-type and vik1Δ cultures each have 22% large budded cells. These strains were also analyzed for growth differences in the presence of benomyl . Unlike vik1Δ mutants, which display increased resistance to benomyl, cik1Δ and cik1Δ vik1Δ mutants are slightly more sensitive than wild-type strains. Therefore, the phenotypic differences of cik1Δ and vik1Δ mutants, combined with their different effects on Kar3p localization, suggest that Cik1p and Vik1p are functionally distinct. The microtubules of the different mutant strains were examined in fixed cells from asynchronous or hydroxyurea-arrested cultures by immunofluorescence with anti-tubulin antibodies. Under these conditions, the microtubules of vik1Δ mutants are indistinguishable from those of wild-type strains. As described previously, cik1Δ mutants have very short spindles compared with wild-type strains and have longer, more abundant cytoplasmic microtubules . This phenotype is identical to that reported for kar3Δ mutants . cik1Δ vik1Δ double mutants display microtubules with no significant difference in length or number to those of cik1Δ mutants (data not shown). Therefore, if disruption of VIK1 results in defects in microtubule structure, these defects are not detected under these conditions. Finally, consistent with the hypothesis that Cik1p and Vik1p function in complexes with Kar3p, the growth rate and microtubule phenotypes of kar3Δ , kar3Δ cik1Δ , kar3Δ vik1Δ , and kar3Δ cik1Δ vik1Δ mutants are all very similar (data not shown). One vegetative function of Kar3p may be to oppose the action of two other S . cerevisiae KRPs, the redundant Cin8p and Kip1p motors . This Kar3p function has been proposed primarily due to genetic interactions between mutations in the genes encoding these KRPs. Disruption of Cin8p and Kip1p function, using the conditional cin8-3 kip1Δ mutant, results in a temperature-sensitive growth defect at 35°C that is partially suppressed by disruption of Kar3p function . However, disruption of CIK1 , encoding a Kar3p KAP, does not suppress cin8-3 kip1Δ , suggesting that the Kar3p activity that opposes Cin8p and Kip1p is independent of Cik1p. Since Vik1p was identified as a second KAP for Kar3p during vegetative growth, we tested whether disruption of Vik1p function could suppress the temperature-sensitive growth defect of the cin8-3 kip1Δ mutant. VIK1 , CIK1 , and KAR3 were deleted individually from the cin8-3 kip1Δ strain, and the resulting mutants were examined for growth defects at 23 and 35°C . As demonstrated previously, the cin8-3 kip1Δ kar3Δ mutant grows significantly better than the original cin8-3 kip1Δ mutant at the restrictive temperature of 35°C . Likewise, disruption of VIK1 also partially suppresses the cin8-3 kip1Δ temperature-sensitive growth defect, as the cin8-3 kip1Δ vik1Δ mutant grows at 35°C. Introduction of a CEN plasmid encoding VIK1 into the cin8-3 kip1Δ vik1Δ mutant restores temperature sensitivity to levels identical to the cin8-3 kip1Δ mutant (data not shown). In contrast, growth of the cin8-3 kip1Δ cik1Δ mutant is significantly diminished at all temperatures compared with the cin8-3 kip1Δ mutant, suggesting a possible added defect in the triple mutant. Therefore, like Kar3p, Vik1p functions antagonistically to Cin8p and Kip1p. This result further indicates that Cik1p and Vik1p are involved in distinct Kar3p functions. Cik1p is a previously described kinesin-associated protein that interacts with the yeast KRP Kar3p . We have characterized a Cik1p-homologous protein called Vik1p that is present in vegetatively growing cells but, unlike Cik1p, is not detected in mating-pheromone treated cells. Coimmunoprecipitation experiments demonstrate that Vik1p also physically associates with Kar3p and that the Kar3p-Vik1p complex is separate from that of Kar3p and Cik1p. Therefore, Kar3p interacts with two different KAPs to form distinct complexes within the same cell. Vik1p requires Kar3p function for its SPB localization. This suggests that Kar3p-Vik1p complex formation and, presumably, the minus-end directed microtubule-motor activity of Kar3p are required to deliver Vik1p to the SPB. In the absence of Kar3p, Vik1p mislocalizes to cytoplasmic patches and is excluded from the nucleus. In contrast, Cik1p mislocalizes throughout the nucleus in the absence of Kar3p during vegetative growth . Two possible models for Vik1p localization can be invoked. First, Vik1p may require association with Kar3p for its nuclear import. In support of this idea, Vik1p does not have sequences predicting a nuclear localization signal. Moreover, Kar3p can target to the nucleus independent of both Cik1p and Vik1p . This nuclear import of Vik1p by association with Kar3p would be analogous to the nuclear import of the yeast γ-tubulin complex. The import of this complex, and its subsequent binding to the nuclear face of the SPB, requires the nuclear localization signal of one of its components, Spc98p . Alternatively, the Kar3p-Vik1p complex may be associated primarily with the cytoplasmic face of the SPB. Therefore, absence of Kar3p would result in release of Vik1p to the cytoplasm specifically, as observed. At this point, we cannot distinguish between these two possibilities, but future studies will address whether the Kar3p-Vik1p complex is on the nuclear, cytoplasmic, or both faces of the SPB. We demonstrate that, whereas Cik1p is required to localize Kar3p to the SPBs and cytoplasmic microtubules of mating pheromone-treated cells , Vik1p is required for proper concentration of Kar3p at the SPBs of vegetatively growing cells. In the absence of Vik1p, Kar3p mislocalizes to nuclear patches, and can be seen along spindle microtubules in many cells. Therefore, Vik1p is required for proper targeting and/or maintenance of Kar3p at the SPB. Vik1p might mediate interactions between Kar3p and other proteins that tether the complex to the SPB. Alternatively, Vik1p could prevent release of Kar3p from the minus-ends of microtubules, where its motor activity would cause it to accumulate. In the absence of both Cik1p and Vik1p, Kar3p again mislocalizes but is more diffuse throughout the nucleus. This suggests that Cik1p may be partially redundant with Vik1p for targeting Kar3p to the SPB. However, Cik1p's primary vegetative function may be to direct localization of Kar3p to other sites within the nucleus, such as nuclear patches and the spindle. This is supported by the observation that the faint nuclear patch staining of Kar3p seen in wild-type cells is diminished in cik1Δ cells. Detection of Kar3p at these sites is greatly enhanced in the absence of its Vik1p-mediated SPB localization. Finally, since some residual SPB staining can be detected in a small percentage of cik1Δ vik1Δ cells, Kar3p may have an inherent KAP-independent ability to localize to the SPB. This localization is likely to depend on its minus-end directed microtubule motor domain, rather than its non-motor stalk and tail domains. These latter regions of Kar3p have been shown previously to be sufficient for its spindle and SPB localization . Cik1p and Vik1p presumably target Kar3p to various sites of action through interactions with its nonmotor domain. Phenotypic, genetic, and biochemical analysis of Kar3p, by several different groups , has strongly suggested that Kar3p is a multifunctional KRP. An intriguing question in the study of molecular motors is how can one motor protein perform several different functions within a single cell.Our results indicate that Kar3p interacts with two related proteins to form three complexes that are involved in distinct microtubule-mediated cellular processes. We believe that the ability of Kar3p to interact with these associated proteins is crucial to its functional versatility. The exact molecular functions of each of these Kar3p complexes are yet to be defined. However, based on phenotypic and genetic analysis, as well as localization studies, our data reveal some possible general roles for the Kar3p-Cik1p complex during mating and the Kar3p-Cik1p and Kar3p-Vik1p complexes during mitosis . The best defined of Kar3p's functions is its role in the nuclear congression step of karyogamy, during which it associates with Cik1p . This complex localizes to cytoplasmic microtubules even in the absence of the Kar3p motor domain , indicating that the nonmotor region of the complex also has microtubule-binding capacity. In the absence of either of these two proteins, microtubules from the SPBs of opposing mating partners fail to interdigitate . Together, these results suggest a model in which the Kar3p-Cik1p complex acts as a cross-linker between antiparallel microtubules emanating from the SPBs of mating partners. The minus-end directed microtubule-motor activity of Kar3p can then create the force that pulls the nuclei together by sliding cross-linked microtubules past one another . Despite many studies on the function of Kar3p during vegetative growth, its role during mitosis remains obscure. Our identification of two Kar3p-interacting proteins with distinct Kar3p-related vegetative phenotypes should help elucidate the exact mitotic functions of this KRP. Disruption of either KAR3 or CIK1 results in similar mitotic phenotypes, including very short spindles indicative of a spindle assembly defect and a mitotic delay mediated by the spindle-assembly checkpoint . Unlike during mating, the Kar3p-Cik1p complex is in the nucleus during the mitotic cell cycle, where it associates with the SPBs and, to a lesser extent, spindle microtubules . Analogous to its role in karyogamy, the complex may act within the spindle to cross-link and slide antiparallel microtubules from opposing SPBs past one another, thereby creating an inward force on the spindle . This force may generate a tension important for proper spindle assembly. Alternatively, the Kar3p-Cik1p microtubule cross-linking activity could be crucial to the organization of a bipolar spindle. This spindle assembly defect would account for the chromosome instability phenotype of cik1Δ mutants . Additionally, it is possible that this complex could play a more direct role in chromosome segregation, perhaps as a kinetochore motor . Kar3p is likely to have a separate mitotic function that is mediated by interaction with Vik1p at the SPBs. The benomyl resistance phenotype of vik1Δ mutants, suggests that the Kar3p-Vik1p complex may be involved in microtubule depolymerization . The motor domain of Kar3p has been shown to possess minus-end–specific microtubule-depolymerizing activity in vitro , and has been suggested by phenotypic analysis to depolymerize microtubules in vivo . Kar3p complexed with Vik1p, specifically, might possess this activity. Alternatively, Kar3p may require interaction with Vik1p to prevent release from microtubule minus ends where it catalyzes microtubule depolymerization. At this point, it is unclear whether this complex acts on cytoplasmic microtubules, the spindle, or both. Disruption of VIK1 does not result in any detectable differences in the microtubule structures of fixed cells compared with wild-type strains. It is possible that accumulation of Kar3p on the spindle, observed in vik1Δ mutants, could stabilize these microtubules and account for the benomyl resistance phenotype. Based on genetic analysis, Kar3p-Vik1p function is detrimental to mutants lacking the plus-end–directed Cin8p and Kip1p KRPs. These proteins are members of the BimC family of KRPs and are believed to act as homotetrameric bipolar motor proteins that generate a SPB separating force during spindle assembly and anaphase B . Disruption of either KAR3 or VIK1 can suppress the temperature-sensitive growth defect of the cin8-3 kip1Δ mutant, suggesting that the Kar3p-Vik1p complex may oppose the function of Cin8p and Kip1p . Interestingly, a TUB2 mutation that stablilizes microtubules can also suppress this mutant , suggesting that a defect in the putative microtubule depolymerizing activity of the Kar3p-Vik1p complex could be sufficient for suppression of cin8-3 kip1Δ . The functional interactions between the two vegetative Kar3p complexes may be complicated. For example, disruption of VIK1 partially suppresses the temperature-sensitive growth defect and mitotic delay of cik1Δ mutants. One interpretation of this result is that the two Kar3p complexes partially oppose one another. It is also interesting to note that cik1Δ mutants, in which Kar3p's SPB localization is unperturbed, have much stronger phenotypes than those of vik1Δ mutants, in which Kar3p is no longer concentrated at the SPBs. Therefore, the most critical of Kar3p functions during vegetative growth may occur at sites other than the spindle poles. Future studies will address these issues, but it is clear from our current study that Cik1p and Vik1p are not functionally redundant. In general, the functional specificity of KRPs is determined by their nonmotor domains. Some KRPs can target to their sites of action independent of associated proteins. For example, the Drosophila Nod protein, involved in chromosome movements during mitosis, contains a DNA binding motif in its nonmotor domain . However, the targeting of many KRPs will likely be mediated through complex formation with nonmotor subunits (i.e., KAPs). The highly divergent nature of the nonmotor domains of KRPs suggest that interacting proteins will also be diverse in sequence. Nevertheless, we expect that mechanisms of motor targeting by KAPs will exist that are universal. The light chains of conventional kinesin are the most studied of all KAPs. Several different KLCs can exist within a single cell , and these are thought to control the cargo-binding specificity of KHCs . Therefore, it is possible that different KLCs target kinesin to distinct membranous organelles and vesicles, and it has been suggested that KLC mediates the interaction between kinesin and membranes . Additionally, KLCs might regulate KHC-microtubule binding and/or motor activity by contacting the motor domain when kinesin is in a folded confirmation . Little is known about the regulation and targeting of specific KRPs. The tail domain of the Xenopus mitotic KRP, Xklp2, has recently been shown to require cytoplasmic dynein and a microtubule-associated protein, TPX2, to localize to spindle poles . However, these proteins are not tightly associated KAPs. Our results demonstrate that Kar3p localization is regulated through its interaction with two different KAPs, Cik1p and Vik1p . These KAPs control various Kar3p functions, at least in part, by targeting the motor to discrete sites within the cell. Whether Cik1p and Vik1p modulate motor activity once Kar3p is at these sites is not yet known. The study of these proteins should define regulatory strategies used by other KRPs and help elucidate general elements underlying the functional diversity of KRPs. | Study | biomedical | en | 0.999995 |
10087266 | LY294002 ( Calbiochem ) was prepared as a stock solution (1,000×) of 20 mM in DMSO and frozen at −20°C. PD98059, 2-(2′-amino-3′methoxyflavone), was purchased from Calbiochem and a stock solution (30 mM; 500×) was prepared in DMSO and frozen at −20°C. The effectiveness of PD98059 was confirmed by its ability to inhibit wound-induced phosphoERK activation. Cycloheximide was prepared as a stock solution (1,000×) of 100 mg/ml in DMSO. The myosin inhibitor 2,3-butanedione monoxime (BDM; Sigma Chemical Co. ) was prepared as a stock solution of 0.5 M in H 2 O and was added to a final concentration of 15 mM and was readded after 3 h. The effectiveness of BDM was assessed by its ability to disrupt the organization of myosin II in stress fibers. The p160ROCK inhibitor, Y-27632 , was prepared as a stock solution of 3 mM in DMSO. The effectiveness of Y-27632 was assessed by its ability to disrupt actin stress fibers and focal adhesions. Primary REF cells were provided by Dr. Durward Lawson (University College London, London, UK). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, streptomycin, and penicillin at 37°C in 10% CO 2 . Wound-induced cell migration assays were performed on secondary cells between passages 3 and 7. Recombinant V14Rho (RhoA isotype), V12Rac (Rac1 isotype), V12Cdc42 (G25K isotype), C3 transferase, N17Rac, N17Cdc42, and WASp (Cdc42 binding fragment comprising amino acids 201–321) were expressed as glutathione S-transferase fusion proteins in Escherichia coli and purified on glutathione-agarose beads essentially as described in Self and Hall . The proteins were released from the beads by thrombin cleavage and dialyzed against microinjection buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl 2 , 0.1 mM DTT) and concentrated as required. For the GTP-binding proteins, active protein concentrations were determined by filter binding assay using [ 3 H]GDP of [ 3 H]GTP as described previously . N17Cdc42 has a low affinity for [ 3 H]GDP and therefore the protein concentration for N17Cdc42 was estimated using a protein assay kit (Bio-Rad Laboratories). The protein concentrations of C3 transferase and the WASp fragment were also determined by this method. Purified neutralizing antibody to Ras, Y13-259, was a kind gift of Dr. Hugh Paterson (Chester Beatty Laboratories, London, UK). REFs for wound assays were seeded at a high density, 12 × 10 4 cells, on 13-mm glass coverslips, and wounded 1 d later when the cells formed a confluent monolayer. The wound was made by scraping a microinjection needle (broken to its shaft and flame polished) through/across the cell monolayer. The wound width was consistently between 100 and 130 μm and wounds reproducibly took between 5 and 6 h to close. Cells were pretreated with inhibitors for 20 min or, in the case of Y-27632, 1 h before wounding. Since most wound edge cells round up immediately upon wounding and thus are difficult to inject, wounds were left for 1 h to allow cell respreading and to facilitate microinjection. Proteins were injected into the cell cytoplasm along with a marker protein (either FITC- or Texas red–conjugated, lysinated dextrans at 2 mg/ml). Recombinant proteins were microinjected at concentrations as indicated in the text. The neutralizing anti-Ras antibody, Y13-259, was microinjected at a concentration of 8–9 mg/ml. Expression vectors (pRK5-myc) encoding N17Rac1, N17Cdc42 (G25K isoform), WASp fragment, and V12HaRas were injected into the cell nucleus at a concentration of 200 μg/ml in PBSA and expressed protein was visualized using anti-myc antibodies (9E10) or in the case of Ras with the rat monoclonal antibody, Y13-238. Previously we have shown that at least 90% of DNA-injected cells express the pRK5 construct and myc-tagged protein could be detected by 30 min after cell injection. For each experimental run, control wounds were fixed soon after the wound was made (for inhibitor experiments), 1 h after wounding (for microinjection experiments) and immediately after the wound edges have met as monitored by frequent observation using phase-contrast optics. Experimental wounds were fixed at the time that control wounds had closed. For wound closure measurements, cells were stained for filamentous actin as previously described . In brief, cells were fixed in 4% paraformaldehyde/1% glutaraldehyde/PBS (in order to preserve fine actin structures such as filopodia), permeabilized in 0.2% Triton X-100/PBS, blocked with sodium borohydride (0.5 mg/ml) in PBS, and stained with rhodamine-conjugated phalloidin (0.1 μg/ml) in PBS. Cells for immunostaining were fixed with 4% paraformaldehyde/PBS, permeabilized, blocked with sodium borohydride, and incubated with primary antibodies diluted in PBS for 1 h at room temperature. Cells were immunostained to reveal vinculin, phosphotyrosine, and myc-tag as described previously . To reveal c-fos, we used a rabbit polyclonal fos antibody (Oncogene Science Inc.) diluted 1:100 followed by FITC-conjugated goat anti–rabbit (1:200; Pierce and Warriner) and a tertiary layer of FITC-conjugated donkey anti–goat (1:200; Jackson ImmunoResearch Labs, Inc.). Dually phosphorylated ERK-1 and -2 were detected using monoclonal anti–MAP kinase, activated and diluted 1:50, followed by FITC-conjugated goat anti–mouse (1:200; Pierce and Warriner) and a tertiary layer of FITC-conjugated donkey anti–goat. Cells were stained for myosin II using affinity-purified rabbit anti–human platelet nonmuscle myosin II antibody (Biomedical Technologies, Inc.) diluted 1:100. To detect expressed Ras protein, cells were permeabilized using 0.1% saponin/80 mM potassium Pipes (pH 6.8)/1 mM MgCl 2 /5 mM EGTA for 2 min before fixation with 4% paraformaldehyde and incubation with rat monoclonal antibodies, Y13-238 (a kind gift of Mike Olson, Chester Beatty Laboratories), followed by Cy3-conjugated donkey anti–rat antibodies (Jackson ImmunoResearch Labs, Inc.). Injected cells were detected with Cascade blue avidin to biotin dextran which was coinjected with the Ras expression vector. Coverslips were mounted by inverting them onto 5 μl movial mountant containing p -phenylenediamine (a few grains/5 ml) as an antifade agent. The coverslips were examined on a Zeiss axiophot microscope using Zeiss 10× and 20× air lenses, and 40× and 63 × 1.4 oil immersion lenses. Fluorescence images were recorded on Kodak T-MAX 400 ASA film. Semiquantitative measurements were made of control wounds ( t = 0) and of control and inhibitor-treated wounds (after the control wound had closed). Three randomly chosen regions of a single wound (each 700 μm long), stained with rhodamine-conjugated phalloidin, were photographed at a magnification of 20. A mean wound width in micrometers was determined (by averaging the width every 30 μm) and an average percent wound closure was calculated. For microinjection experiments, a region of wound was randomly chosen and a continuous stretch of 10–15 leading edge cells on opposing sides of the wound was microinjected. Since not only front row cells participate in the wound response, we also injected cells in rows 2, 3, and 4 on both sides of the wound. For microinjected wounds, wound width was measured as the distance between microinjected cells (on either side of the wound) visualized by coinjected fluorescent dextran or by anti-myc staining, since some noninjected cells often crawl between injected cells into the wound. In this case, measurements were made every 15 μm over a length of wound of ∼300–400 μm. From the average values for each injection, percent wound closure was calculated. To record the position of the Golgi apparatus in migrating wound edge cells, wider wounds (∼200 μm wide) were made to monolayers of REFs and cells were fixed 2, 4, and 6 h after wounding. The Golgi apparatus was localized by immunolabeling, as described above, using a rat monoclonal antibody (23C) which recognizes β-COP. The position of the immunolabeled Golgi apparatus in each cell was recorded as described in Kupfer et al. . Wound edge cells were divided up into three 120° sectors centering on the nucleus, one of which faced the edge of the wound . Cells in which the Golgi apparatus (i.e., 50% or more of the fluorescent image) was within the sector facing the wound were scored positive and for each time point at least 100 cells were examined. For the zero time point the random value of 33.3% of cells showing Golgi apparatus in the forward facing sector was given. To examine the effect of mutant GTPases on Golgi apparatus reorientation, wounds were injected with expression vectors encoding myc-tagged N17Rac, N17Cdc42, WASp, and V12Cdc42 1 h after the wound was made. Expressed myc-tagged proteins were detected 1 h later when 50% of cells repositioned their Golgi apparatus to the forward facing sector. The cells were fixed 5 h later and Golgi apparatus position was recorded in wound edge cells expressing myc-tag. A CCD camera and time-lapse controller (EOS Electronics) were attached to a Zeiss inverted microscope. Injected or inhibitor-treated cells were placed in a slide flask . Microscope images were collected at a rate of 10 frames every 60 s on a Sony betacam video recorder. Individual frames were transferred from videotape to a Macintosh computer with a frame grabber and processed using Adobe Photoshop. After time-lapse, cells were fixed and stained to identify microinjected cells. To investigate the role of small GTPases in fibroblast motility, small scrape wounds were made across a confluent monolayer of primary REFs . The wound width is approximately two to three cells across (mean 120 μm) and during a period of 5–6 h, cells move bodily forwards to fill the gap without the necessity for cell division . The response to wounding is rapid; within 30 min, rounded and retracted cells at the wound edge respread and extend filopodia and lamellipodia into the open space. 2 h after wounding, front row cells have clearly developed a polarized morphology, with lamellipodia and membrane ruffling localized only at the leading edge and not at the sides or rear edge of cells, where they are in contact with neighboring cells . The wound edge cells remain fairly tightly packed and adherens junctions, visualized with cadherin antibodies, remain intact during the course of the assay (data not shown). Rhodamine-conjugated phalloidin reveals that all cells in the monolayer contain abundant stress fibers, but in addition, cells at the leading edge also have actin-rich lamellipodial and filopodial extensions . Antiphosphotyrosine antibodies reveal elongated focal adhesions in all cells and cells at the leading edge also have smaller focal complexes intimately associated with lamellipodia and filopodia . After ∼5 h, as opposing cells approach each other, time-lapse cinematography reveals that cell ruffling ceases within minutes of cell–cell contact (data not shown). Wounding results in the rapid induction of various transcription factors such as c-fos and Egr-1 in cells both at the wound edge and several rows back into the monolayer . We observe c-fos upregulation in four or five rows of cells back from the leading edge (data not shown). However, inhibition of protein synthesis, by pretreatment of the cells with cycloheximide (100 μg/ml), has no effect on wound closure (percent wound closure is 90 ± 3.6; control = 96 ± 2.9%) and we conclude that changes in gene transcription and protein synthesis are not required for forward movement in this short-term assay. Cell motility is generally associated with the protrusion of two types of actin-rich structure, lamellipodia and filopodia, at the leading edge of cells and in Swiss 3T3 fibroblasts, these are controlled by Rac and Cdc42, respectively . To determine whether Rac plays a role in wound-induced cell motility, patches of cells on opposite sides of a wound were microinjected with either dominant negative Rac (N17Rac) protein, or with an expression vector encoding N17Rac. Rhodamine-conjugated phalloidin staining revealed that inhibition of Rac blocked all lamellipodial activity . Cells were left with a “scalloped” shape, although filopodia could still be seen . Inhibition of Rac had severe effects on cell movement and quantitative analysis revealed that in cells injected with N17Rac protein wound closure was blocked by ∼80%, whereas in cells injected with the N17Rac expression construct cell movement was inhibited by 98%. The difference in these values likely reflects the instability of N17Rac protein . Microinjection of constitutively active Rac protein (V12Rac) had no inhibitory effect on cell movement . We conclude that Rac is activated upon wounding and that Rac activity is essential for cell movement. To determine whether Cdc42 also plays a role in wound closure, patches of cells on opposite sides of a wound were injected with either dominant negative Cdc42 (N17Cdc42) protein, or with an expression vector encoding N17Cdc42. It can be seen from Fig. 3 that inhibition of Cdc42 results in a 50% inhibition of cell movement. Microinjection of cells with the Cdc42-binding domain of the Cdc42 target protein, WASp, resulted in a similar level of inhibition , confirming that Cdc42 is required for efficient movement but is not absolutely essential. To examine the role played by Cdc42 during wound closure, we looked more carefully at migrating cells lacking Cdc42 activity. Fig. 4 A shows the typical polarized morphology of a wound edge cell (visualized with injected fluorescent dextran) with ruffling activity restricted to the front edge. In contrast, Fig. 4 B shows the morphology of a wound edge cell in which Cdc42 has been inhibited. Cell polarity is completely lost and protrusive, lamellipodial activity can be seen all around the cell's periphery. Expression of constitutively active Cdc42 (V12Cdc42) protein had no effect on cell polarity or forward movement . The polarized morphology of migrating cells also involves the reorganization of the microtubular network and the alignment of the Golgi apparatus in the direction of movement . Although no major reorganization of the microtubular network could be seen during the REF wound healing assay (data not shown), disruption of microtubules with nocodazole did inhibit wound closure by 60 ± 6% ( n = 3). Realignment of the Golgi apparatus, on the other hand, could be clearly observed during the assay. As seen in Fig. 5 , A and B, during the course of the experiment the percentage of cells with their Golgi apparatus orientated in the direction of movement rose from 33% (essentially random) to >80%. Although the relatively long time course suggests that this realignment is not essential for movement, it may facilitate movement and is a clear read-out of a developing polarized morphology. To determine whether Cdc42 activity is required for this aspect of cell polarity, Golgi apparatus reorientation was measured in wound edge cells after injection with dominant negative Cdc42 or with the WASp fragment. Fig. 5 (C–E) shows that inhibition of Cdc42 completely prevents Golgi apparatus realignment. Wound-induced Golgi apparatus reorientation was not inhibited by constitutively active Cdc42 (V12Cdc42) or by dominant negative Rac (N17Rac) . In Swiss 3T3 fibroblasts, Rho is required for both the formation and maintenance of actin stress fibers and focal adhesions . REF monolayers have abundant stress fibers, and after wounding motile edge cells continue to display many actin stress fibers oriented both towards the wound and radially across the cell . To determine whether these structures are required for cell movement, cells were microinjected with the Rho inhibitor, C3 transferase, at a concentration of 65–75 μg/ml. As shown in Fig. 6 , B and C, at this concentration of C3 transferase, cells are devoid of all visible stress fibers and focal adhesions, yet they continue to extend lamellipodia and filopodia normally and wounds close . Although Rho activity, as visualized by stress fiber formation, is not required for wound closure, microinjection of C3 transferase at a fourfold higher concentration (300 μg/ml) results in loss of substrate adhesion and severe cell retraction (data not shown) and under these conditions wound closure is significantly inhibited . Using this experimental approach, we were unable, therefore, to assess whether the absence of actin stress fibers/focal adhesions could enhance cell movement, since inhibition of Rho with C3 transferase has at least two distinct effects. However, stress fibers and focal adhesions can be inhibited more directly by blocking the activity of one of the downstream targets of Rho, p160ROCK , with compound Y-27632. In the presence of 20 μM Y-27632, wound edge cells contained few actin stress fibers (data not shown), but remained attached to the coverslip . Under these assay conditions, there is a significant increase in the speed of wound closure by ∼30% as indicated by the position of the wound edges in the presence and absence of Y-27632. Finally, microinjection of a constitutively active Rho protein (V14RhoA), which is unable to cycle through an inactive GDP-bound state, severely inhibits wound closure . Injection of V14Rho does not obviously increase the number of actin stress fibers in wound edge cells, which are already abundant, although in some cases stress fibers and focal adhesions appear to be thicker than in control cells (data not shown). We conclude that Rho-dependent stress fibers and focal adhesions are not required for cell movement, but that a basal Rho activity is required to maintain cell substrate adhesion as previously described . Furthermore, focal adhesions and stress fibers must undergo dynamic turnover during movement and this requirement results in an inhibitory effect on the speed of movement. Ras has been proposed to play a role in regulating the turnover of focal adhesions . To determine whether Ras is activated during the assay, wounded cells were visualized with an antibody that recognizes dually phosphorylated ERK, a downstream target of the Ras pathway. Fig. 7 shows that ERK is activated rapidly after wounding (within 5 min), but that the levels decrease to background by 1 h. Interestingly, ERK activation is seen to extend further back from the wound edge than c-fos induction (eight rows as opposed to only four). Wound-induced ERK activation can be blocked by microinjection of the neutralizing Ras antibody, Y13-259 (data not shown), or by preincubation with the MEK inhibitor , indicating a requirement of the Ras/ MEK pathway for wound-induced ERK activation. To determine whether Ras is required for cell movement, patches of cells around a wound were microinjected with the neutralizing Ras antibody Y13-259 . Inhibition of Ras has no effect on lamellipodia protrusions , nor does it affect cell polarity (data not shown). Nevertheless, cell movement is inhibited by 80% . Further analysis by time-lapse videomicroscopy of cells in which Ras has been inhibited revealed normal protrusive activity, but no net cell movement. However, we did observe that actin stress fibers and focal adhesions in the Ras-inhibited cells appeared to be somewhat thicker and larger than those in control cells, similar to what we observed in cells injected with constitutively active Rho. These observations suggested a possible link between Ras and focal adhesion and stress fiber turnover. To test this, cells were injected with the Ras inhibitor Y13-259 and were either coinjected with the Rho inhibitor C3 transferase, or were treated with the p160ROCK inhibitor Y-27632. Under both conditions, stress fibers and focal adhesions were lost as expected (data not shown), but now cells migrated normally resulting in wound closure . Interestingly, when V12Ras is expressed at relatively low levels and at early times (2 h) after microinjection of an expression construct, we clearly observe Ras concentrated at focal adhesion sites using the anti-Ras antibody, Y13-238 . After longer times (6 h), V12Ras has a significant affect on cell morphology, inducing loss of adhesions and changes in cell shape, typical of a transformed morphology. Ras regulates a number of signaling cascades including the ERK-MAP kinase pathway and in some, but not all, cells PI3-kinase . To examine whether either of these two activities might be responsible for the Ras requirement seen here, we have used the MEK inhibitor PD98059 and the PI3-kinase inhibitor LY294002. Either of these inhibitors alone results in a relatively weak inhibition of wound closure and even in combination, wound closure is only inhibited by ∼50%. We conclude that neither of these downstream pathways is likely to account for the specific Ras requirement during wound closure. The analysis of a model cell line, Swiss 3T3 fibroblasts, has led to the identification of three signal transduction pathways responsible for controlling the organization of the actin cytoskeleton and each is regulated by a member of the Rho family of small GTPases . Rho promotes the assembly of actin–myosin contractile filaments (stress fibers) and associated focal adhesion complexes, Rac induces the formation of protrusive lamellipodial structures , while Cdc42 triggers filopodia formation . Since the assembly and disassembly of filamentous actin structures drive cell movement, axon guidance, phagocytosis, and cytokinesis, it is likely that Rho GTPases will play key roles in controlling these fundamental biological processes. Here we examine the individual contributions of Rho, Rac, and Cdc42 to cell movement. Animal cells move by crawling along a surface and this involves a combination of protrusive activity at a leading edge combined with forward movement of the nucleus and cell body . Cells may move either as individuals or as a group, often in sheets, and examples of both types of movement can be found in the embryo and in the adult. To analyze how cells move cooperatively, we have used an in vitro wound healing assay. Individual cells in a confluent fibroblast monolayer are nonmotile and appear to have low levels of Rac and Cdc42 activity since only a few, sporadic membrane protrusions can be seen. Rho, on the other hand, appears to be highly active, since cells display many stress fibers and focal adhesions. Wounding of the monolayer induces cells at or close to the leading edge to crawl forward to close the gap and during the 5-h course of our experiments, the cells move cooperatively as a sheet, retaining close contacts with their neighbors. The most obvious and immediate effect of wounding is to induce (within minutes) dynamic activity, lamellipodial and filopodial protrusions, and membrane ruffling, at a leading edge. This is consistent with a wound-induced activation of Rac and Cdc42, although it is formally possible that these GTPases are already active in the monolayer, but functionally silent due to cell–cell contacts. In any event, microinjection of wound edge cells with a Rac inhibitor prevents lamellipodia formation and membrane ruffling (filopodia are still seen) and there is no forward movement over the time course of the experiment. Others have reported that Rac is required for chemotaxis of fibroblasts towards PDGF and macrophages towards CSF-1 , for hepatocyte growth factor–induced scattering of MDCK epithelial cells , and for the invasive movement of some metastatic cells . Furthermore, genetic disruption of Rac in the Drosophila embryo prevents the actin cytoskeleton changes required to drive dorsal closure, a process involving the movement of sheets of epithelial cells around the early embryo . It appears, therefore, that Rac may be universally required to drive cell crawling, consistent with its unique role in controlling the formation of lamellipodia. The other protrusive structures seen after wounding are filopodia and, as expected from work with Swiss 3T3 cells, these can be completely blocked by inhibition of Cdc42 in wound edge cells. However, this results in only a partial block (∼50%) of wound closure and it appears that although Cdc42 activity is required for efficient cell movement, in this assay it is not absolutely essential. Nevertheless, closer inspection of cells in which Cdc42 has been inhibited reveals an extremely interesting role for this protein. Normally, cells at the wound margin develop a morphological polarity showing a clear leading edge (with membrane ruffles and filopodia), but no protrusional activity at their sides or rear, where they are in contact with neighboring cells. In addition, migrating cells polarize their Golgi apparatus to a position forward of the cell nucleus and in the direction of migration and disruption of the Golgi apparatus directly with brefeldin A, or indirectly with nocodazole (to disrupt microtubules) inhibits directed cell migration . We observed that inhibition of Cdc42 in wound edge cells caused a complete loss of polarity; cells protrude lamellipodia all around their periphery, irrespective of whether there are cell–cell contacts, and Golgi apparatus reorientation does not occur. We conclude that Cdc42 is not required to activate Rac, but that it is required to restrict Rac activity to the leading edge through the generation of a polarizing signal. These observations point to some interesting analogies with the yeast Saccharomyces cerevisiae , where Cdc42 is required for the formation of the bud during cell growth . Inactivation of Cdc42 causes a disruption of the actin cytoskeleton and a loss of cell polarity and directed secretion; as a consequence, cell growth becomes isotropic . These results bare striking similarities with what we observe in the wound assay. However, one significant difference is the effect of a constitutively active Cdc42; in yeast this also causes loss of cell polarity but in wound edge cells it has no obvious effect on their polarity. Perhaps the presence of cell–cell contacts in fibroblast monolayers provides an additional constraint, and in this respect, it is interesting to note that both dominant negative and constitutively activated Cdc42 prevent directed cell migration, though not motility per se, of isolated macrophages in a chemotaxis assay and reorientation of the MTOC in isolated T cells after interaction with antigen-presenting cells . Our observations point to some interesting interplay between Cdc42 activity and cell–cell contacts. The mechanism by which Cdc42 generates polarity in multicellular organisms is unknown, although in yeast, Cdc42 is directed to the bud site by Bud1, another small GTPase in the Ras family. It is not even clear whether filopodia are required or whether they are formed after polarity has been established. Our experiments suggest two extreme roles for Cdc42: (a) it directs protrusion (i.e., Rac activity) and secretion (microtubule-dependent Golgi apparatus reorientation) to the leading edge; or (b) it is required for inhibition of protrusive activity from regions involved in cell–cell contact. By analogy with yeast, we favor the first model but we cannot exclude the second. Contractile actin–myosin filaments are found in all animal cells; in cultured fibroblasts these are readily seen as well organized bundles (stress fibers), tethered to the plasma membrane at specialized sites (focal adhesions) . The REFs used in the wound healing assay have abundant stress fibers and focal adhesions and these are maintained even in leading edge cells after wounding. It is often said that these structures likely inhibit locomotion , and we have confirmed this using the Y-27632 compound, an inhibitor of the Rho-effector p160ROCK which mediates actin stress fiber assembly . Y-27632 induces a loss of actin stress fibers and focal adhesions (without obviously affecting cell shape or attachment) and causes a significant (30%) increase in the rate of cell migration. Interestingly, the myosin ATPase inhibitor BDM, which also stimulates loss of actin stress fibers, inhibited cell motility by 30% (data not shown), suggesting that other contractile, actin–myosin filaments may play some role during cell movement in this assay. Several groups have identified an essential role for Ras in cell movement, both in chemotaxis and in wound closure assays . We also find that inhibition of Ras blocks cell movement, but we also show that movement can be fully restored by simultaneous inhibition of focal adhesion/stress fiber assembly by inhibiting either Rho or its effector protein, p160ROCK. This experiment strongly suggests that Ras regulates the turnover of focal adhesions and stress fibers, clearly an important feature of migrating cells . This could occur in at least two ways. First, Ras might regulate the Rho GTPase cycle. Since Rho is required for the formation of focal adhesions, their turnover may depend on Rho inactivation (i.e., formation of Rho.GDP from Rho.GTP), which is likely to be controlled by a GTPase-activating protein (GAP). One obvious candidate molecule that could provide a link between the two GTPases is p120RasGAP, which is known to exist in a complex with p190RhoGAP, a GAP active on Rho. In fact, overexpression of the NH 2 -terminal, non-GAP containing region of p120RasGAP leads to loss of stress fibers . The second possibility is that Ras regulates focal adhesion turnover independently of Rho. In support of this model is the observation that although the p160ROCK inhibitor Y-27632 restores cell movement after Ras inhibition, it does not restore cell movement in cells expressing constitutively activated Rho protein (V14Rho) (data not shown). However, the mechanism by which Ras might regulate focal adhesion turnover is not clear. Inhibition of MEK or PI3-kinase (two downstream pathways controlled by Ras) appears to have only minor effects on movement. This is in contrast to previous reports suggesting that PI3-kinase and the MAP kinase pathway are important for motility . Furthermore, the components of the focal adhesion complex that are the target of Ras-mediated signals are also not clear, although focal adhesion kinase is one molecule that has been suggested to play an important role in their turnover . In conclusion, we have shown that Rac is essential for producing the leading edge protrusions required for forward movement in a wound healing assay. Cdc42 activity is essential to maintain the polarized phenotype of migrating cells and Rho is required for cell adhesion. Ras is also essential for promoting focal adhesion turnover during migration. Major outstanding questions concern the nature of the biochemical pathways that mediate these GTPase functions and how they are coordinated in time and space. | Study | biomedical | en | 0.999998 |
10087267 | Actin was purified from rabbit muscle and isolated as Ca-ATP-G-actin by Sephadex G-200 chromatography in G buffer (5 mM Tris-Cl − , pH 7.8, 0.1 mM CaCl 2 , 0.2 mM ATP, 1 mM DTT, 0.01% NaN 3 ). Actin was pyrenyl-labeled or rhodamine-labeled on lysines in the F form as described . Profilin was purified from bovine spleen as described . VASP bacterial recombinant protein was expressed and purified as described . For recombinant Evl protein, a fragment encoding amino acids 1–393 of Evl was generated via PCR and cloned in pFB-Nhis ( GIBCO-BRL ). Recombinant protein was obtained and purified using the Bac-to-Bac™ baculovirus expression system ( GIBCO-BRL ) according to the manufacturer's instructions. Recombinant baculovirus-infected high-five insect cells were harvested and the recombinant protein purified by affinity to Talon resin ( Clontech ) according to the manufacturer's instructions. The coding region of Evl was amplified for a cDNA using the following primers: forward primer: 5′-Cgg gAT CCA TgA gTg AAC AgA gTA TCT gC-3′, reverse primer: 5′-Cgg Cgg CCg CCg Tgg TgC TgA TCC CAC-3′. GST fusion proteins of EVH1 domains of Mena or Evl have been described elsewhere . Fragments encoding the EVH1 domain of VASP (amino acids 1–149) or the proline-rich repeat region of ActA (amino acids 241–423) were generated by PCR, and cloned into the pGEX2TK vector ( Pharmacia ). Prokaryotic recombinant GST-EVH2 (amino acids 226–380) was obtained by generating the fragment from full-length VASP by PCR and cloning it into the pGEX4T1 vector ( Pharmacia ). The GST fusion proteins were expressed and purified following manufacturer's instructions. The monoclonal VASP antibody (clone IE245) used in this study was described recently . The mAb against Evl (clone 84H1) was raised against the GST fusion protein of Evl-EVH1, and specificity was confirmed on pure Evl-EVH1 protein after thrombin digestion of the GST fusion protein. Both antibodies are IgGs and have been purified by affinity chromatography using protein G–Sepharose. The VASP polyclonal antibody M4 was purchased from Immunoglobe. The (GP 5 ) 3 peptide of the VASP proline-rich region was synthesized with an AMS 222 Multiple Sequencer using TentaGel S resin (Rapp Polymere). The creation and analysis of the Mena knockout mice is described in Lanier et al. . The complete human VASP cDNA was PCR amplified from a clone obtained at Genome System Inc., using the following two oligonucleotide primers: 5′-GCG ATA AGG ATC CGa tga gcg aga cgg tca tct g-3′ and 5′-GCG CCT GGT ACC tca ggg aga acc ccg ctt cc-3′. The pBlueBacHis2B-VASP plasmid was generated by ligating the BamH1/Kpn1 double digest PCR fragment with the BamH1/Kpn1 digested pBlueBacHis2 B (Invitrogen BV). The resulting polyhistidine-enterokinase site NH 2 -terminal–tagged VASP nucleotide sequence was verified by DNA sequencing. Generation of the VASP recombinant baculovirus was achieved by using the standard procedure of transfection–recombination in Sf21 insect cells . Positive virus clones were identified by Western blot analysis using both the M4 anti-VASP rabbit pAb or the anti-Xpress™ mAb (Invitrogen BV). Sf21 insect cells were cultured in Grace's supplemented media ( GIBCO-BRL ) containing 10% FBS ( GIBCO-BRL ) and 0.001% pluronic acid ( GIBCO-BRL ) in 1 liter polycarbonate Erlenmeyer flask at 27°C under vigorous shaking. 250 ml of cell suspension (1.5–2.5 × 10 6 /ml) was infected with the recombinant VASP baculovirus at a multiplicity of infection ranging from 2 to 5 for 60 h. Harvested infected Sf21 cells were rinsed once in ice-cold PBS and disrupted on ice in 10 ml of lysis buffer (20 mM sodium phosphate, pH 8.0, 5% glycerol, and 1 mM DTT) containing protease inhibitors (0.5 mM PMSF, 10 μg/ml benzamidine, 5 μg/ml chymostatin, and 5 μg/ml leupeptin). The mixture was centrifuged for 20 min at 45,000 g at 4°C. The supernatant was kept on ice. The pellet was resuspended in 10 ml of buffer A (20 mM sodium phosphate, pH 8.0, 300 mM NaCl, 5% glycerol, 0.03% Tween 20, 1 mM DTT, and protease inhibitors), homogenized in a Dounce potter, and left on ice for 30 min before a 20-min centrifugation run at 45,000 g at 4°C. The supernatant was added to the first one and the whole solution was dialyzed against 500 ml of 20 mM sodium phosphate, pH 8.0, 5% glycerol, 0.03% Tween 20, and 1 mM DTT (two changes). The cellular extract containing VASP was rapidly drop-frozen in liquid nitrogen and kept at −80°C. Purification of the insect recombinant VASP was achieved at 4°C by a two-step chromatography procedure. The cellular extract was laid on a 10-ml DEAE-cellulose matrix DE52 (Whatman) equilibrated with buffer B (20 mM sodium phosphate, pH 8.0, 0.5 mM DTT, 5% glycerol, and 0.03% Tween 20) and the protein flow-through was collected. NaCl was added to this protein fraction up to a 50 mM final concentration before mixing with 500 μl of TALON™ metal affinity resin ( Clontech ) equilibrated with buffer C (20 mM sodium phosphate, pH 8.0, 50 mM NaCl, 5% glycerol, and 0.5 mM DTT) for 30 min. The suspension was deposited on an empty column and the flow-through was discarded. The resin was rinsed with 20 ml of buffer C and VASP was eluted with buffer D (20 mM sodium phosphate, pH 7.0, 50 mM NaCl, 0.5 mM DTT, 5% glycerol, 100 mM imidazole, pH 7.0, 100 mM EDTA, pH 7.0, and protease inhibitors). VASP was dialyzed against VASP buffer (20 mM Hepes-NaOH, pH 7.8, 30 mM NaCl, 1 mM DTT, and 5% glycerol). The composition of VASP buffer was worked out to optimize the solubility of VASP. After centrifugation at 400,000 g at 4°C for 30 min, the protein was stored at −80°C at a concentration of 10–20 μM. The concentration of VASP was determined by the Bradford protein assay (Bio-Rad) using BSA as a standard. 2–3 mg of pure VASP was thus obtained from a 250-ml culture. Electrophoretic patterns of the eukaryotic recombinant protein showed two bands migrating as 54- and 50-kD polypeptides, representing 40 and 60% of the material, respectively. Both polypeptides were recognized by the anti-VASP M4 antibody and the anti-Xpress mAb (Invitrogen). After treatment with PKA catalytic subunit, the material migrated as a single 54-kD polypeptide, and after treatment with alkaline phosphatase, as 50 kD. Therefore, the two bands of the eukaryotic recombinant VASP represent the serine 157–phosphorylated VASP (49.5 kD) and the unphosphorylated VASP (46 kD) usually found in platelets , to which the 4-kD fusion peptide has been added. Platelet extracts were prepared from outdated unstimulated human platelets essentially as described . The platelet lysate obtained by sonication in buffer A containing 10 mM Tris-Cl − , pH 7.5, 10 mM EGTA, 2 mM MgCl 2 , and antiproteases (10 μg/ml of leupeptin, pepstatin, and chymostatin, and 1 mM PMSF) was centrifuged at 100,000 g for 45 min at 4°C. The supernatant was supplemented with 2 mM ATP, 2 mM DTT, and 150 mM sucrose, rapidly frozen on liquid nitrogen, and stored at –80°C. Mouse brain extracts were prepared by homogenization of brains from freshly decapitated mice in two volumes of ice-cold buffer A, using a 0.5 ml glass/Teflon potter (20 strokes), followed by sonication (3 × 15 s, low power) and centrifugation at 100,000 g for 1 h at 4°C. The supernatant was frozen on liquid nitrogen and stored at −80°C. L . monocytogenes strain Lut12 (pactA3) overexpressing ActA was used. Bacteria were grown overnight at 37°C in brain/heart infusion medium in the presence of 7 μg/ml chloramphenicol and 5 μg/ml erythromycin. The culture was then brought into fresh medium (5 ml) at an optical density of 0.3 at 560 nm, and grown to stationary phase (1.5–2.0 OD units at 560 nm). After addition of 30% glycerol to the medium, bacteria were frozen in 100-μl aliquots on liquid nitrogen and stored at −80°C. Frozen bacteria were thawed, centrifuged, and resuspended at 6 × 10 9 bacteria/ml in B buffer (10 mM Hepes, pH 7.7, 0.1 M KCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 , and 50 mM sucrose). The motility assay solution (16 μl) was prepared at room temperature by mixing 4 μl of cytoplasmic brain or platelet extract and 12 μl additional ingredients (from concentrated stock solutions) coming up to the following final concentrations: 0.38% methylcellulose, 5 μM F-actin (added to platelet extracts only), 6 mM DTT, 2 mM ATP, 4 mM MgCl 2 , 1.5 × 10 −3 % DABCO as an oxygen scavenger, 1 μM rhodamine-actin, and 2 × 10 8 bacteria/ml. After thorough mixing, 2.5 μl of the suspension was squashed between a BSA-coated slide and a 20 × 20-mm coverslip. The depth of the chamber was 6 μm. The sample was sealed with VALAP (Vaseline/lanosterol/paraffin, 1:1:1). Movement of L . monocytogenes was observed at room temperature 10 min after preparation of the sample in a Zeiss III RS microscope equipped with a Lhesa LHL4036 silicon-intensified camera. Bacteria were observed both in phase-contrast and in fluorescence. Movement was recorded in real time or time lapse using a Panasonic video recorder. Rates of movement were measured using a Hamamatsu image analyzer. 6–15 measurements were made on different bacteria for each sample. Average rates were derived from the distances moved over a period of 1 min. Standard deviations were calculated using the statistical tools of Kaleidagraph software. Many bacteria (>200) were counted to derive the percentage of motile bacteria in a given experiment. All results shown have been obtained reproducibly in at least four independent experiments, and data shown in figures are typical data. VASP was immunodepleted from platelet extracts using 4.5-μm-diameter goat anti–mouse IgG-coated magnetic Dynabeads ( Dynal ). The binding capacity was 0.3–0.4 μg antibodies per 10 7 beads. 2 × 10 8 Dynabeads were suspended and extensively washed in PBS containing 1 mg/ml BSA, then incubated with a sixfold excess of monoclonal anti-VASP antibody (mAb 245) which was shown, by epitope mapping, to recognize the proline-rich central region of VASP. The concentration of the antibody was 5.8 mg/ml. Incubation was carried out for 30 min at room temperature, with rotary stirring. After extensive washing of the beads with PBS containing 0.1% BSA, the beads were split in two batches for two consecutive cycles of depletion. In the first cycle 10 8 anti-VASP–coated beads were incubated with 20 μl of platelet extract at 4°C for 90 min with rotary stirring. The supernatant from the first cycle of depletion was submitted to a second cycle using the second batch of Dynabeads. Mock-depleted extracts were obtained by identical treatment of extracts by uncoated Dynabeads. The extent of VASP deletion was quantitated by immunoblotting using a polyclonal anti-VASP antibody. Evl immunodepletion from mouse brain extracts was performed as follows. 4 × 10 8 Dynabeads were coated (to saturation) with monoclonal anti-Evl for 30 min at room temperature and split into four batches. The supernatants were removed. Four consecutive cycles of depletion of 20 μl of brain extracts were performed at 4°C for 60 min. Mock-depleted extracts were treated identically in parallel using uncoated Dynabeads. Profilin was depleted from platelet extracts by poly- l -proline chromatography. 50 μl of extract was chromatographed through 50-μl poly- l -proline-agarose columns that had been first equilibrated in 10 mM Tris-Cl − , pH 7.5 buffer, then rapidly centrifuged in a bench Eppendorf centrifuge to remove interstitial buffer. The extract-loaded columns were centrifuged at 4°C for 1 min. The profilin-depleted flow-through was recovered. This operation was repeated once to obtain >98% depletion of profilin , as checked by immunoblotting. The extract thus obtained appeared to be partially depleted (∼60%) in VASP. After a third chromatography step on poly- l -proline-agarose, VASP was no longer detected by immunoblotting. Since the lowest detectable amount of VASP is 20 nM and the total amount of VASP in platelet extracts is 0.7 μM (see Results), we consider that the extent of depletion of VASP was at least 97%. This last result also demonstrates that VASP binds to the poly- l -proline column. VASP-induced polymerization of MgATP-G-actin was monitored at 20°C by the increase in fluorescence of pyrenyl-labeled actin using a Spex Fluorolog 2 spectrophotometer. The excitation wavelength was 366 nm, and the emission wavelength was 407 nm. The excitation and emission slits were maintained small enough so that the fluorescence measured at 407 nm was not contaminated by scattered light due to bundle formation. Alternatively, the polymerization process was monitored by turbidimetry using a Cary 1 spectrophotometer, at a wavelength of 310 nm and using 120-μl cuvettes thermostatted at 20°C. At the end of the polymerization process, samples were centrifuged at 400,000 g for 20 min at 20°C. The amounts of sedimented F-actin and unassembled actin were determined by SDS-PAGE of the resuspended pellets and of the supernatants. Gels were either Coomassie blue stained or silver stained according to Morrissey . The gel patterns were scanned using an Arcus (Agfa) scanner and analyzed using the Image Analysis NIH software. Samples of preincubated MgATP-G-actin (1 μM) and VASP (1–2 μM) were deposited on air glow-discharged carbon-coated grids, negatively stained with 2% uranyl acetate, and observed in a Philips CM12 electron microscope. Electron micrographs were taken at a 35,000-fold magnification. VASP was immunodepleted from platelet extracts using magnetic Dynabeads coated with an antibody directed against the proline-rich region of VASP. Listeria did not move in VASP-depleted extracts, whereas the mock-depleted extracts fully supported motility as efficiently as the untreated extracts. Specifically, the fraction of motile bacteria (30–40%) was the same in the untreated and mock-depleted extracts. Although no actin tails were assembled around Listeria after depletion of VASP, actin actively polymerized at the surface of the bacteria into large, loosely organized, veil-like actin meshworks, as illustrated in Fig. 1 . Add-back of the bacterial recombinant VASP protein at concentrations as low as 0.25 μM restored the movement of bacteria. The optimum concentration of VASP was 0.5–1 μM. Data showing the extent of VASP depletion and the dependence of the speed of bacteria on VASP concentration are displayed in Fig. 2 . The speed of propulsion was not affected by addition of 1 μM VASP to the undepleted extracts. Upon addition of larger amounts of VASP (5 μM), bright actin bundles of rhodamine-F-actin were observed in platelet extracts and inhibition of movement started to be observed. Using recombinant VASP as a standard, the concentration of VASP in platelet extracts was determined to be 0.7 ± 0.2 μM by immunoblotting , which corresponds to ∼4 ± 1 μM in the platelet cytoplasm, assuming a sixfold dilution of the cytoplasm in the extracts . The successful reconstitution of Listeria movement was observed with unphosphorylated bacterial VASP as well as with the eukaryotic recombinant VASP in an identical range of concentrations . VASP has been shown to interact with ActA via its NH 2 -terminal EVH1 domain. To verify the implication of this interaction in Listeria movement, extracts were supplemented with 2.5 μM GST-EVH1 fusion proteins of VASP, Mena, and Evl which share sequence homology (Table I ). No inhibition of Listeria movement was observed when bacteria were added to the extracts supplemented with GST-EVH1. On the other hand, when bacteria were separately preincubated with GST-EVH1 proteins for 20 min before being added to the extracts, so that the final concentration of GST-EVH1 was again 2.5 μM, bacteria initially did not move and appeared surrounded by disorganized actin clouds. After 30 min of incubation in the extracts, bacteria slowly resumed normal movement and comet tails eventually were seen. The rate of propulsion eventually decreased at late times, most likely due to ATP depletion. These data indicate that the EVH1 domain of VASP, Mena, and Evl is in slow association–dissociation equilibrium with the poly- l -proline region of ActA. In addition, the fact that endogenous VASP at 0.7 μM eventually displaces ActA-bound EVH1 when isolated EVH1 is present at 2.5 μM indicates that the affinity of VASP in the macromolecular assembly is much higher than the affinity of EVH1 for ActA, suggesting that VASP interacts either with other regions of ActA or with partners other than ActA at the surface of Listeria , and these interactions strengthen its overall affinity for ActA. To discriminate between the above two possibilities, the effect of the GST- (proline-rich region of ActA) fusion protein on Listeria motility was tested. Addition of the fusion protein at concentrations as low as 0.8 μM (a concentration equimolar to endogenous VASP) completely blocked the movement of Listeria , and reduced by twofold the percentage of bacteria able to initiate assembly of actin at the bacterium surface. This result demonstrates that the proline-rich region of ActA binds VASP with high affinity, and the VASP-Pro(ActA) complex cannot further interact with ActA. Accordingly, the movement of Listeria was not reconstituted in VASP-depleted extracts when bacteria were preincubated with GST-EVH1 proteins. In conclusion, VASP binds ActA via interaction of the EVH1 domain of VASP with the proline-rich region of ActA, and this interaction is necessary but not sufficient for the activity of VASP in motility. Hence the interaction of VASP with other ligands increases the overall affinity of VASP for the macromolecular assembly responsible for motility, and is necessary for actin-based motility of Listeria . Overall, the sequence of the Evl protein is most similar to VASP, in particular the EVH1 domains of VASP and Evl present 61% identity. The Evl recombinant protein was found able to restore actin-based motility of Listeria in VASP-depleted platelet extracts . Interestingly, the rate of movement was lower upon addition of Evl to the VASP-depleted extracts than upon addition of VASP. Since the EVH1 domains of VASP and Evl are equally able to bind ActA, this difference may be due to differences in the interaction of VASP and Evl with another partner. The role of Mena and Evl in Listeria movement was addressed using Mena knockout (−/−) animals. Mouse brain extracts prepared as described in Materials and Methods were assayed for Listeria movement. Images of Listeria undergoing actin-based motility in brain extracts are displayed in Fig. 3 . The rate of propulsion was 5–10-fold slower than in platelet extracts (0.15–0.3 μm/min). Because of these very slow rates, the standard deviations in rate measurements were as high as 30–40%. For that reason, the motility data in brain extracts were not reliably expressed in terms of rates and the statistics were preferably expressed in terms of percentages of motile bacteria and of bacteria surrounded by actin “clouds” but not moving. In contrast to the observation made on platelet extracts, the rate of movement did not increase, but decreased, upon addition of exogenous F-actin to the motility assay mixture. Therefore, motility assays were performed without additional F-actin. Preliminary experiments showed that the percentages of motile bacteria (all motile bacteria formed comet tails) and of bacteria surrounded by actin clouds were identical in brain extracts from wild-type animals and from knockout (both heterozygous −/+ and homozygous −/−) animals, leading to the tentative conclusion that the Evl protein, homologous to Mena, is sufficient to support movement in the absence of Mena. Quantitative immunoblotting assays show that the 48-kD Evl protein is present at a concentration of 0.7 μM in brain extracts. Identical amounts of Evl were found in brain extracts from wild-type and Mena (−/−) animals (data not shown). Immunodepletion of Evl in wild-type or heterozygous Mena (+/−) extracts did not affect the movement of Listeria . The rates of movement were the same as in the undepleted controls (within the 30% standard deviation). In contrast, partial immunodepletion of Evl in Mena (−/−) extracts (not shown) caused an appreciable inhibition of movement and of the formation of regular actin tails. A large proportion of coiled, disorganized actin comets was then seen around the bacteria. Complete inhibition of movement and a total absence of actin tails were obtained upon depletion of Evl in Mena (−/−) extracts . Since the lowest detectable amount of Evl is 50 nM (see below), the extent of depletion was at least 93%. However, polymerization of actin in unorganized meshworks was still observed around the bacteria. Hence Evl, like VASP, was not required for actin polymerization per se. Add-back of 1.1 μM pure recombinant Evl to Evl-depleted extracts of Mena (−/−) mice restored Listeria motility. Motility was equally well restored by addition of 0.6 μM bacterial recombinant VASP. The percentage of motile bacteria in the add-back of Evl or VASP was twice lower than in the mock-depleted control. Despite this lower performance, which was less quantitatively observed in other independent experiments, the difference between the Evl-depleted samples, which are totally immotile, and the (depleted + add-back of Evl or VASP) sample was reproducible and testifies for the role of Evl or VASP in movement. In conclusion, the requirements for Listeria motility in Evl-depleted brain extracts of Mena (−/−) mice were identical to the ones observed in VASP-depleted platelet extracts. Addition of a twice larger amount of VASP led to the massive formation of large bundles of rhodamine-actin in the brain extract, and subsequent inhibition of the formation of actin tails. These results show that Mena, Evl, and VASP play interchangeable roles in Listeria movement and most likely bind to the same molecular targets in this function. Depletion of profilin in platelet extracts by poly- l -proline chromatography did not abolish the movement of Listeria . In the total absence of profilin, and 60% of endogenous VASP, obtained by two consecutive poly- l -proline chromatography steps of the platelet extracts (see Materials and Methods), Listeria moved at a 30% lower speed than in the mock-depleted extract. Double depletion of both VASP and profilin by three consecutive poly- l -proline affinity columns (see Materials and Methods) caused the arrest of movement. Add-back of profilin alone did not restore movement. Movement was restored in the double-depleted extracts by adding back VASP alone (60% of the bacterial speed observed in the mock-depleted extract). Further addition of profilin increased the rate of movement to 80% of the value observed in the mock-depleted extract . These results demonstrate that profilin is not required for Listeria motility, in agreement with previous results obtained in Xenopus egg extracts , and that VASP can support Listeria movement independent of profilin. The profilin-induced 30% increase in speed, an indication that profilin increases the efficiency of motility, may be due to profilin's known function in actin dynamics (see Discussion), or to its ability to interact directly with the (GP 5 ) sequence, repeated to different extents in VASP/Mena/Evl proteins. To further examine the latter possibility, the effect of the (GP 5 ) 3 peptide on Listeria movement in platelet extracts was assayed. Addition of the peptide at 1 mM slowed down the movement to an extent that varied widely from 20 to 60% in a large number of different experiments. The result contrasts with the very efficient inhibition of movement which was observed when micromolar amounts of this peptide were injected in infected cells . A similar weak and low-affinity effect was obtained upon addition of poly- l -proline ( Sigma Chemical Co. ) at concentrations (5–10 mM proline) comparable to those of (GP 5 ) 3 . Since VASP itself binds to poly- l -proline (see Materials and Methods), we cannot eliminate the possibility that the (GP 5 ) 3 peptide and poly- l -proline inhibit movement by displacing VASP from the proline-rich region of ActA as weak competitors. The low-affinity inhibitory effect of the (GP 5 ) 3 peptide on Listeria movement contrasts with the very efficient inhibition observed by addition of micromolar amounts of EVH1 or Pro-ActA fusion proteins. In summary, no evidence for a functional role of the central region of VASP in Listeria movement can be drawn from the effect of this peptide in the present in vitro assay. Many tissues contain two or all three proteins (Mena, VASP, Evl) of the Ena/VASP family . Since these proteins play interchangeable roles in Listeria movement, one can legitimately wonder why the VASP present in Evl-depleted Mena (−/−) mouse brain extracts or the Evl protein present in VASP-depleted platelet extracts does not support Listeria movement. One possible explanation could be that the amounts of soluble Evl in platelets and of VASP in brain are so low that after depletion of the major variant (VASP in platelets, Evl in Mena (−/−) brains), the concentration of the less abundant protein is below the threshold at which movement can be observed. To test this possibility, the amounts of Evl present in platelet extracts and of VASP in brain extracts were evaluated by quantitative immunoblotting. Fig. 6 shows that when the highest possible volumes of extracts (obtained with low ionic strength buffer, in the absence of detergent) were loaded on the gel, VASP could not be detected in brain extracts, nor Evl in platelet extracts. Considering the lower limits of immunodetection of these proteins, the result indicates that the concentration of soluble Evl in platelet extract is lower than 50 nM, and the concentration of soluble VASP in brain extracts is lower than 20 nM. These amounts appear too low to support movement of Listeria . Hence, our results suggest that the fraction of soluble Mena and Evl is larger than the fraction of soluble VASP in brain under the lysis conditions used to prepare these extracts. Experiments using higher ionic strength/ detergent lysis indicate that pools of VASP in brain and Evl in platelets exist which are not solubilized by these conditions . The above results demonstrate that VASP, Evl, and Mena play interchangeable roles in actin-based motility of Listeria . They bind ActA via their EVH1 domain and their common function implies that they interact with another unknown common ligand. Like EVH1, the EVH2 domain is well conserved among the Ena/VASP proteins. Truncation of this region abolishes the localization of VASP to focal contacts , indicating that this region, like EVH1, may play a role in the targeting of VASP, though the direct contacts with ActA or zyxin are likely to be mediated only through the EVH1 domain. Since VASP appears necessary, in Listeria motility, to transform actin polymerization in “clouds” into an organized actin structure supporting active movement, we assayed the interaction of recombinant VASP with actin by in vitro assays combining pyrenyl-actin fluorescence, light scattering, electron microscopy, and sedimentation measurements. Such in vitro assays allow a quantitative comparison of the nonphosphorylated bacterial VASP recombinant protein with the partially phosphorylated eukaryotic recombinant VASP. Both recombinant proteins induced the polymerization of G-actin at low ionic strength, suggesting that VASP binds polymerized actin preferentially. The effect of VASP was very similar to the myosin-subfragment-1–induced assembly of G-actin , except that it was ATP insensitive. The polymerization process of MgATP-G-actin induced by eukaryotic recombinant VASP was monitored by the increase in fluorescence of pyrenyl-labeled actin and by the increase in light scattering or in turbidity at 310 nm . Very similar, if not strictly kinetically identical data were obtained with the bacterial recombinant protein (data not shown). While the extent of pyrenyl fluorescence and the shape of the fluorescence emission spectrum were compatible with the formation of F-actin, the large increase in turbidity indicated that particles much larger than actin filaments were assembled from G-actin in the presence of VASP. The extent of fluorescence change varied with the concentration of VASP as a high-affinity titration curve , indicating that 1 mol of actin was assembled per mole of VASP monomer added, which was confirmed by sedimentation assays . In contrast, the extent of turbidity change did not display a saturation behavior, because the thickness of the bundles increases with protein concentration and with time, resulting in a nonlinear concentration and time dependence of the turbidity. Electron microscope observation of negatively stained specimens showed that thick F-actin bundles were formed with bacterial and eukaryotic recombinant VASPs. The formation of bundles is fully consistent with the turbidity data. VASP-induced polymerization of G-actin was observed even at submicromolar concentrations of VASP and actin, consistent with the high affinity of VASP for F-actin at low ionic strength displayed in Fig. 7 a, inset. Polymerization was much more efficient from Mg-actin than from Ca-actin. The induction of actin assembly in filament bundles by VASP was favored in low ionic strength buffers (<20 mM KCl) and was slowed down upon increasing the ionic strength . The formation and ionic strength dependence of F-actin bundles upon addition of VASP are both consistent with the change in charge of filaments after VASP binding and the existence of electrostatic forces between filaments in the bundles . To examine which region of VASP is involved in actin binding, the effect of the EVH2 domain, which contains the highly charged COOH-terminal region of VASP, was investigated. The GST-EVH2 fusion protein of VASP induced F-actin assembly like VASP, except that no bundles were formed . The absence of bundles when EVH2 was bound to F-actin indicates that the overall charge of EVH2-F-actin is different from that of VASP-F-actin. Competition between VASP and GST-EVH2 was observed . The interaction of VASP with F-actin was then examined in vitro as follows. Preliminary sedimentation assays first showed that both bacterial and eukaryotic recombinant VASP cosedimented with F-actin (1–5 μM) in a 1:1 molar ratio of monomeric VASP per F-actin subunit, confirming the conclusions of the experiments of induction of actin assembly. The fluorescence of pyrenyl-labeled F-actin was not affected by the binding of VASP. Fig. 8 a shows that in a low ionic strength buffer (MgATP-actin, 15 mM NaCl, 0.1 mM MgCl 2 added to G buffer), addition of 1 μM VASP shifted the critical concentration for F-actin assembly from 0.2 to 0.1 μM, indicating that VASP increased the stability of F-actin, i.e., bound F-actin preferentially. The affinity of VASP for F-actin under physiological conditions (0.1 M KCl, 1 mM MgCl 2 ) was estimated using a sedimentation assay. The assays were performed using standard F-ADP-actin and F-ADP-BeF 3 -actin, which mimics the F-ADP-Pi state of F-actin, in parallel samples. The rationale for this experiment was to determine whether VASP, in being bound to ActA and to the barbed ends of growing filaments, which have F-ADP-Pi terminal subunits, would show a functionally relevant preference for either one of the F-ADP or F-ADP-Pi conformations of F-actin. No difference was observed in the binding of bacterial and eukaryotic recombinant VASPs to F-ADP and F-ADP-Pi (data not shown). On the other hand, the two eukaryotic VASP polypeptides corresponding to the serine 157–dephosphorylated form (46 kD, which we will call dephospho-VASP) and the serine 157–phosphorylated form (50 kD, which we will call phospho-VASP) bound F-actin with different affinities. Typical data displayed in Fig. 8 b show that when 0.5 μM eukaryotic VASP was mixed with 1 μM F-actin, phospho-VASP, which represented ∼30% of the material, was practically all bound to F-actin, consistent with an equilibrium dissociation constant lower than 0.05 μM, while 50% of dephospho-VASP was bound to F-actin, consistent with an equilibrium dissociation constant of ∼1 μM. The values of the equilibrium dissociation constants for binding of dephospho-VASP and phospho-VASP to F-actin, K X , and K Y , respectively, were derived from the quantitative analysis of the scanned gel patterns using a model in which the two forms of VASP bind to the same site on F-actin. The following equations were used: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{K}}_{X}=[{\mathit{F}}]/([{\mathit{X}}_{0}]/[{\mathit{X}}]-1)\end{equation*}\end{document} with [ X 0 ] = 0.7 [ V 0 ] and [ Y 0 ] = 0.3 [ V 0 ]. [ V 0 ], [ X 0 ], and [ Y 0 ] represent the total concentrations of VASP, of dephospho-VASP, and of phospho-VASP, respectively; [ X ] and [ Y ] represent the concentrations of free dephospho- and free phospho-VASP measured in the supernatants; and [ F ] represents the concentration of unliganded F-actin subunits, derived from the difference between total F-actin and the sum of F-actin–bound phospho- and dephospho-VASP, measured in the pellets. Values of 0.8 ± 0.1 and 0.02 ± 0.01 μM were thus found for K X and K Y . In conclusion, phosphorylated VASP binds F-actin with a 40-fold higher affinity than dephosphorylated VASP. It has been demonstrated that the shift in apparent molecular mass of VASP from 46 to 50 kD is linked to phosphorylation of serine 157 exclusively, while phosphorylation at other sites (serine 239, threonine 278) does not affect the electrophoretic migration in denaturing gels . Accordingly, both the 46-kD and the 50-kD polypeptides may be partially phosphorylated on other sites, e.g., serine 239 . Nonetheless, in its high-affinity binding to actin, the 50-kD phospho-VASP polypeptide behaves as a homogeneous population, which suggests that the phosphorylation of serine 157 is solely responsible for the strong binding to F-actin. A different, more complex, bimodal behavior would be displayed in the binding curves if phosphorylation of serine 239 affected the affinity of VASP for F-actin. In conclusion, the phosphorylation of serine 157 by the cAMP-dependent protein kinase regulates the affinity of VASP for F-actin. Our results showing that VASP binds F-actin are in qualitative agreement with earlier data using VASP purified from platelets , but the binding stoichiometry and the affinity of VASP for actin were not estimated, and the experiments were not conducted under conditions appropriate to measure a difference in affinity between phosphorylated and nonphosphorylated VASP. Finally, cosedimentation assays, displayed in Fig. 8 , c and d, showed that GST-EVH2 bound to F-actin in a 1:1 molar ratio with an equilibrium dissociation constant of 0.7 μM and in competition with VASP. In conclusion, the interaction of VASP with F-actin is mediated by its EVH2 domain. To understand the function of the interaction of VASP with F-actin in motility of Listeria , the effect of the GST-EVH2 on Listeria movement in platelet extracts was tested. Addition of 3.5 μM GST-EVH2 to the motility assay mix led to the formation of ill-defined actin tails. Upon addition of 7 μM GST-EVH2, no actin tails were formed and bacteria did not move. These data, which corroborate the competition between VASP and EVH2 for binding to F-actin, indicate that the interaction of VASP with actin, mediated by the EVH2 domain, is essential in its function in actin-based motility. The fact that a 10-fold molar excess of EVH2 over VASP is necessary to inhibit movement, while the affinity of EVH2 and of VASP for F-actin are identical, is again consistent with the multiple interaction of VASP in the macromolecular assembly responsible for motility. The in vitro motility assay of L . monocytogenes in platelet and brain extracts has been used as a tool to demonstrate that proteins of the Ena/VASP family are required for actin-based motility. Since active actin polymerization, but no movement, is observed around the bacteria after selective removal of VASP from platelet extracts (or of Mena and Evl from brain extracts), we conclude that these proteins do not act as actual nucleators of actin assembly, but as organizers of the actin meshwork for the efficient production of propulsive force. This conclusion is in full agreement with the one derived from genetic studies showing that the deletion of the four proline-rich repeats of ActA caused a severe inhibition of Listeria movement in infected cells and in Xenopus egg extracts . Although bacteria expressing modified ActA still induced actin assembly, the number of bacteria moving (albeit at a fourfold reduced speed) was 10-fold lower than for wild-type bacteria. A 5.5-fold reduced speed of movement in infected cells was also observed in a second report . The quantitative difference between the observations derived from genetically modified Listeria and the present in vitro VASP/Mena/Evl depletion studies lies in the fact that in the previous experiments, proteins of the Ena/VASP family were still present in the medium. Therefore, the possibility cannot be discounted that low-affinity binding of these proteins to subsites on ActA, undetectable by immunofluorescence, would be sufficient to elicit slow movement of a few bacteria. Interestingly, a motility phenotype similar to the one obtained upon VASP removal (actin polymerization around the bacteria, indicating that Arp2/3 was recruited, but no actin tail formation and no movement) was observed upon deletion of the 117-KKRRK-121 peptide in the NH 2 -terminal region of ActA . Although VASP was bound to these NH 2 -terminal deletion ActA mutants, the phenotype suggests that it may not have been bound in a functional fashion. In summary, while the proline repeats of ActA may represent the main VASP binding subsite, other regions of ActA may also be involved in VASP function. VASP, Mena, and Evl appear to play interchangeable roles in actin-based motility, in different cellular contexts. Evl can replace VASP in VASP-depleted platelet extracts, and VASP can replace both Mena and Evl in Mena knockout, Evl-depleted brain extracts. This conclusion is in agreement with a recent report showing that, in vivo, human VASP can substitute for the loss of Ena in the Drosophila embryo. It shows that these proteins are likely to interact with the same targets in different cells, and that the role of Ena in neural development may be, in analogy with the function of VASP in Listeria movement, to organize the growth of filaments at the plasma membrane for the correct development of neural cells. Whether VASP, Ena, Mena, and Evl are similarly regulated in different tissues is an open question. Ena/VASP proteins clearly can support Listeria movement independently of profilin, since the simple add-back of VASP to extracts that have been double-depleted from VASP and profilin is sufficient to restore movement of Listeria at a rate which is 75% of the rate observed upon add-back of both VASP and profilin. Listeria movement does not require profilin, however the increase in bacterial speed provided by profilin is consistent with its function in filament turnover . In the presence of ADF (2 μM in platelet extracts), profilin increases the turnover of actin filaments, which powers the movement of Listeria . Whether an additional effect is provided by the interaction of VASP/Mena with profilin is an issue which is not elucidated by our work. The engineering of mutated forms of VASP unable to bind profilin but binding ActA and F-actin in an unaltered fashion, or of mutated forms of profilin unable to bind poly- l -proline but interacting with actin only, in a complex able to participate in barbed end assembly like unmodified profilin appears required to clarify this point. In this context, it is noteworthy that a recent report studying the role of profilin in N-WASP–elicited filopodia extension in response to EGF demonstrates that the ability of profilin to interact directly with actin is required for N-WASP–mediated filopodial extension, and that mutations in the proline-rich region of N-WASP which abolish the binding of profilin to N-WASP did not impair filopodium extension, although the length of the microspikes was greatly reduced. The in vitro motility assay of Listeria provides quantitative estimates of the affinities of the different domains of the Ena/VASP proteins involved in building the molecular scaffold responsible for actin assembly and movement. The recombinant VASP and Evl proteins used in this work were efficient at submicromolar concentrations. Use of the GST-EVH1 and GST-(Pro-ActA) fusion proteins of VASP, Mena, and Evl demonstrates that the EVH1 domain itself binds the ActA proline-rich region in slow association–dissociation equilibrium, and with an affinity which, given the very slow dissociation rate, we tentatively estimate at least in the 10 7 M −1 range. This implies that the attachment of VASP to ActA is quasi-permanent during the movement of Listeria . VASP interacts also with actin via its EVH2 domain. The isolated EVH2 domain binds F-actin in a 1:1 molar ratio and an affinity of 0.7 μM, identical to the binding stoichiometry and affinity of dephospho-VASP for F-actin. The fact that EVH2, in displacing VASP from F-actin, abolishes the movement, demonstrates that the interaction of VASP with the filaments of the actin tail is essential in propulsion. In binding its two targets, ActA and actin, VASP may work as a molecular connector linking the surface of the bacterium to the growing filament via its EVH1 and EVH2 domains. This result is in full agreement with the strong bonding between the bacterium and the actin tail measured with optical tweezers (Gerbal, F.B., V. Laurent, A. Ott, P. Chaikin, M.-F. Carlier, and J. Prost, manuscript in preparation). Our results expand the conclusion of these mechanical studies in showing that the attachment of the actin tail to the bacterium is required for movement, a conclusion at variance with the original Brownian ratchet model . One can imagine that the connection elicited by VASP between ActA and actin imposes a structural constraint in the arrangement of the elongating barbed ends at the surface of Listeria , maintaining them attached to the bacterium and bundled in a defined orientation, which would allow the development of force in privileged location and direction. In the absence of VASP, the growing filament ends are randomly oriented, leading to the formation of disorganized actin clouds from which a productive force against the bacterium wall cannot result. The EVH2 domain of Ena has been proposed recently to be involved in multimerization of Ena . In view of the present data showing the interaction of EVH2 with actin, it is also possible that F-actin was present as a third partner serving to mediate or stabilize the interaction. More detailed investigations will have to be carried out to address this possibility. The present data lead us to formulate the following model. During Listeria movement, VASP remains located at the bacterium–actin tail interface, implying that while the EVH1 domain is bound to ActA, the EVH2 domain slides along the side of the barbed end of the growing filament, allowing insertional polymerization of actin. This working model of a molecular ratchet is illustrated in Fig. 9 . Within this model, in maintaining the site of filament growth at a defined location and distance from the bacterium surface, each added subunit would contribute in pushing the bacterium, hence VASP would increase the yield of the transformation of actin polymerization into propulsive force. Since statistically all filaments do not attach and detach simultaneously, considering the collective behavior of the population of filaments assembled at the bacterium surface is essential in understanding the mechanism of movement. Factors controlling the strength of VASP interaction with actin, i.e., the frequency of attachment–detachment steps, are expected to regulate the filament sliding rate, hence to control the speed of propulsion of Listeria . The fact that the affinity of VASP for F-actin is increased 40-fold (up to 0.5 × 10 8 M −1 ) by phosphorylation of serine 157 raises the possibility that cAMP-dependent phosphorylation of VASP slows down the movement of Listeria . This possibility is currently under investigation. Our data and conclusions compare well with those derived from a recent study showing that VASP-null platelets aggregate faster. This observation suggests that the role of VASP, which would be enhanced by phosphorylation, could be in part, via actin binding, to maintain the cohesive integrity of the membrane actin cytoskeleton of unstimulated platelets. In conclusion, the use of selectively depleted platelet and brain extracts provides a useful tool for elucidating the role of Ena/VASP family proteins in Listeria motility and in normal cellular function. | Study | biomedical | en | 0.999998 |
10087268 | All mice ( Jackson Laboratories ) used were male C57-black-10smsc-DMD- mdx mice and control littermates aged 6 mo. Animals were anesthetized by intraperitoneal injection of a combination of ketamine (80 mg/kg) and xylazine (7 mg/kg). Fixed tissue was obtained by perfusing the anesthetized animal through the left ventricle with a solution of 2% paraformaldehyde in PBS (10 mM NaP, 145 mM NaCl, pH 7.2). Fast twitch muscles, including the tibialis anterior, extensor digitorum longus, and quadriceps muscles, were dissected; the quadriceps muscle was incubated for an additional 5 min in 2% paraformaldehyde in PBS. Tissue was blotted dry and placed on a cryostat chuck in O.C.T. mounting medium . The chuck was plunged into a slush of liquid nitrogen, generated under vacuum. 20-μm-thick sections were cut on a cryostat , collected on glass slides coated with 0.5% gelatin and 0.05% chromium potassium sulfate, and stored desiccated at −70°C. For the preparation of unfixed longitudinal sections, tissue was obtained without perfusion, mounted, frozen, and cryosectioned as above. To prevent contraction, the frozen sections were collected on the surface of an ice-cold bath of PBS containing 10 mM EGTA, pH 7.4. Sections were rapidly lifted from the surface of the bath directly onto gelatin-coated slides. Unfixed longitudinal sections were cut fresh for each experiment and used immediately. Mouse mAbs (Novocastra Laboratories) to the COOH terminus of human dystrophin (Dys2; ref. 54), to the COOH terminus of human β-dystroglycan ( 5 ), and to a fusion protein containing the NH 2 -terminal region of utrophin (NCL-DRP-2; ref. 4) were used at dilutions of 1:5, 1:10, and 1:5, respectively. An mAb against all known syntrophin isoforms, 1351, was provided by Dr. S. Froehner (University of North Carolina, Chapel Hill, NC), and was used at 16 μg/ml. Mouse mAbs ( Affinity Bioreagents ) against the α subunit of the dihydropyridine receptor (DHPR 1 ; clone 1A), and the sarcoplasmic/endoplasmic reticulum Ca-ATPase from rabbit skeletal muscle (SERCA 1) were used at dilutions of 1:200 and 1:100, respectively. Mouse mAbs against α-actinin from rabbit skeletal muscle (EA-53) and vinculin from chicken gizzard (VIN-11-5; both from Sigma Immuno Chemicals) were used at dilutions of 1:200 and 1:50, respectively. The rabbit polyclonal antibody, 9050, was prepared against purified human erythrocyte β-spectrin. It was affinity-purified over a column of erythrocyte β-spectrin and cross-adsorbed against β-fodrin and α-fodrin, purified from bovine brain as previously described ( 63 , 87 ). Antibodies to erythrocyte β-spectrin were also generated in chickens. IgY was purified from egg yolk using the EggStract kit ( Promega Corp. ) and anti–β-spectrin antibodies were affinity-purified as described ( 63 ). Specificity for β-spectrin was demonstrated by immunoblotting (Ursitti, J.A., L. Martin, W.G. Resneck, T. Chaney, C. Zielke, B.E. Alger, and R.J. Bloch, manuscript submitted for publication). Both affinity-purified antibodies to β-spectrin were used at 3 μg/ml. Polyclonal rabbit antibodies to a fusion protein containing residues 335–519 of the α1 subunit of the Na,K-ATPase and to a fusion protein containing residues 338–519 of the α2 subunit of the Na,K-ATPase (Upstate Biotechnologies) were used at 3 μg/ml. Nonimmune mouse mAbs, MOPC21, were obtained from Sigma Chemical Co. Nonimmune chicken IgY was purified from preimmune controls collected from the same chickens and used to generate the anti– β-spectrin antibodies. Normal rabbit serum was purchased from Jackson ImmunoResearch Laboratories. Secondary antibodies included goat anti–rabbit and goat anti–mouse IgGs, and donkey anti–chicken IgY. All secondary antibodies (Jackson ImmunoResearch) as fluorescein or tetramethylrhodamine conjugates were species-specific with minimal cross-reactivity and were used at a dilution of 1:100. The specificities of all these antibodies have been established by immunoblotting or immunoprecipitation, as reported by the suppliers or in the relevant publications. Sections were incubated in PBS/BSA (PBS containing 1 mg/ml BSA and 10 mM NaN 3 ) for 15 min to reduce nonspecific binding and placed in primary antibody in PBS/BSA for 2 h at room temperature, or overnight at 4°C. Samples were washed with PBS/BSA and incubated for 1 h with fluorescein– or tetramethylrhodamine–conjugated secondary antibodies diluted in PBS/BSA. After additional washing, samples were mounted in a solution containing nine parts glycerol, one part 1 M Tris-HCl, pH 8.0, supplemented with 1 mg/ml p -phenylenediamine to reduce photobleaching ( 37 ). Slides were observed with a Zeiss 410 confocal laser scanning microscope ( Carl Zeiss, Inc. ) equipped with a 63× NA 1.4 plan-apochromatic objective. The pinholes for both fluorescein and tetramethylrhodamine fluorescence were set to 18. Images were collected and stored with software provided by Zeiss . To generate figures, images were arranged into montages, labeled, and given scale bars with Corel Draw 6 (Corel Corporation Ltd.). Inset pictures were prepared with MetaMorph ( Universal Imaging ) and magnified twofold with Corel Draw 6. No other processing was used on any of the images. For the quantitations shown in Fig. 3 , images were prepared by one of the authors and scored double blind by the other. Images were taken of all sarcolemmal regions in controls and mdx myofibers that did not show obvious tears, holes, or other processing artifacts. Sampling was otherwise random. Images of control and mdx sarcolemma were coded and mixed randomly. Each image was then evaluated for the pattern of β-spectrin distribution that was most common over the region of the sarcolemma in clear focus. Images were sorted into one of four categories: clear costameric distribution, including regular labeling over Z and M lines and in longitudinal domains; label present at Z lines, but absent over M lines or in longitudinal domains; label present at Z lines but absent both over M lines and in longitudinal domains; and label absent at Z lines, M lines, and longitudinal domains, but present in polygonal arrays or other irregular structures. We obtained consistent results when other naive observers scored the same or a similar set of images. Unless otherwise stated, all materials were purchased from Sigma Chemical Co. and were the highest grade available. We used immunofluorescence labeling and confocal microscopy to examine the distribution of several membrane skeletal proteins in longitudinal sections through the sarcolemma of fast twitch muscle fibers from the dystrophic mdx mouse. The use of confocal optics allowed us to isolate the fluorescence signal arising from proteins located within ∼1.2 μm of the sarcolemmal bilayer ( 83 ), thereby reducing any fluorescence signals, specific or nonspecific, associated with structures lying deeper in the myoplasm. Each of the sarcolemmal proteins we examined has been shown to be concentrated in costameres, together with dystrophin and β-spectrin (12, 32, 47, 49, 59, 62, 73; Williams, M.W., and R.J. Bloch, manuscript in preparation). We limited our observations to fast twitch fibers because the rectilinear pattern generated by immunolabeling of membrane skeletal elements at the sarcolemma, overlying Z and M lines and in longitudinally oriented strands, is much more clearly defined than in slow twitch fibers (Williams, M.W., and R.J. Bloch, manuscript in preparation). In addition, fast twitch fibers are more susceptible to damage associated with dystrophinopathies ( 82 ). Longitudinal cryosections were prepared from the quadriceps muscle of control and mdx mice. Sections from control and mdx samples were placed on the same slides and were treated simultaneously during all stages of our experiments. We first labeled sections with 9050, an affinity-purified polyclonal antibody against β-spectrin, and Dys2, an mAb against dystrophin. Primary antibodies were visualized with species-specific secondary antibodies coupled to either fluorescein or tetramethylrhodamine. In controls, β-spectrin was found in costameres, areas overlying the Z lines and M lines, and in longitudinally oriented strands , as we reported previously (62; see also 12). In our samples, costameres overlying Z and M lines could be distinguished under fluorescence illumination by the fact that structures over Z lines were usually wider than those over M lines ( 62 ). Labeling of all these structures was specific, as it was not obtained in either wild-type or mdx tissue with nonimmune rabbit antibodies or with a secondary antibody that failed to bind rabbit IgG. Dystrophin, recognized by labeling with Dys2, was also enriched in costameres , as previously reported ( 47 , 49 , 62 , 73 ). The distribution of β-spectrin in the quadriceps of the mdx mouse differed significantly from that in control muscles. Although in some cases the β-spectrin distribution was very similar to that seen in the control tissue such examples were rare in mdx muscle (<3% of 108 fibers scored double blind). Many of the samples (∼36%) failed to show labeling for β-spectrin either over M lines or in longitudinally oriented strands . Approximately the same proportion of mdx fibers (∼42%) failed to show labeling of both these structures , and so retained organized domains of β-spectrin only over Z lines. This pattern of labeling is similar to that reported by Porter et al. ( 62 ), Minetti et al. ( 50 ), and Ehmer et al. ( 20 ). The mdx muscle fibers that could not be placed into any of the above categories showed different morphologies, including small irregular boxes, zig-zag patterns, or polygonal arrays . Some regions of the sarcolemma of mdx muscle lacking visible β-spectrin extended over several sarcomeres or encompassed large areas (>50 μm 2 ) of the sarcolemma between Z lines . The fact that all of these patterns were associated with muscle fibers that lacked dystrophin was confirmed by double immunofluorescence studies with anti– β-spectrin and Dys2 anti-dystrophin. The latter antibody did not label any structures at the sarcolemma of the dystrophic muscle fibers studied here (not shown). In contrast to our results with mdx muscle fibers, we found that >75% of control myofibers had regular rectilinear distributions of β-spectrin at the sarcolemma , with prominent elements in longitudinal strands and overlying Z and M lines. Most of the remaining control fibers were not labeled at the sarcolemma either over M lines or in longitudinal domains. Only one control fiber out of the 72 that we scored did not show labeling of the sarcolemma over M lines or longitudinal domains. We found no control fibers with irregular boxes, polygonal arrays, or zig-zag patterns as seen in mdx . Typically, the regions of the control sarcolemma that failed to label with antibodies to β-spectrin extended over areas of only 1–2 μm 2 (the area enclosed by adjacent Z and M lines and a pair of well-spaced longitudinal strands) in control muscle fibers. In very few cases (<1% of all fibers in controls and mdx ), we found regions of the sarcolemma of muscle fibers that showed little or no organization of the sarcolemma. These may represent regions that were poorly preserved or damaged during processing. The altered arrangement of proteins at the sarcolemma of dystrophic muscle fibers was not due to differences in fixation or labeling of these fibers. We routinely examined samples that were fixed by perfusion in situ before the muscle was dissected and cryosectioned, as well as samples that were unfixed and collected under conditions that prevented contraction following cryosectioning (see Materials and Methods). To examine their possible effects, we also varied the perfusion and fixation regimens. We found no significant differences among these samples, suggesting that our results were independent of the methods we used. Furthermore, control and mdx samples were matched for age and sex, so these factors did not contribute to the differences in sarcolemmal organization. Finally, most control and mdx samples were also collected, frozen, sectioned, and stained together. Therefore, this ruled out the possibility that differences in handling could account for the morphological changes we observed. These results suggest that the organization of β-spectrin at the sarcolemma of mdx is altered from the control. The differences between the mdx and control samples, summarized in Fig. 3 , are highly significant . Unfixed longitudinal cryosections of quadriceps muscle from control and mdx animals were labeled in double immunofluorescence protocols for β-spectrin, with either 9050 or chicken anti–β-spectrin antibodies, and with antibodies to other integral membrane or cytoskeletal proteins, including syntrophin, β-dystroglycan, vinculin, utrophin, and the α1 and α2 subunits of the Na,K-ATPase. Structures labeled by primary antibodies were visualized with species-specific secondary antibodies coupled to either fluorescein or tetramethylrhodamine. The α1 and α2 subunits of the Na,K-ATPase form a complex with ankyrin and β-spectrin at the sarcolemma of skeletal muscle fibers (Williams, M.W., and R.J. Bloch, manuscript in preparation). This results in the codistribution of the two subunits of the Na,K-ATPase with β-spectrin in costameres . Therefore, we expected both subunits to redistribute with β-spectrin at the sarcolemma of mdx muscle. This prediction was confirmed by double immunofluorescence experiments . We observed extensive overlap in the distributions of these proteins with β-spectrin . The absence of labeling at regions overlying M lines, the disruption of longitudinally oriented strands and structures overlying Z lines, and the presence of irregular polygonal structures were evident in the patterns of labeling of the α1 and α2 subunits of the Na,K-ATPase, as they were for β-spectrin. β-Dystroglycan is the transmembrane glycoprotein of the dystrophin-associated glycoprotein complex that binds dystrophin ( 39 , 77 ). Syntrophin is a peripheral membrane protein that binds to dystrophin at sites COOH-terminal to those recognized by β-dystroglycan ( 2 , 9 ). In control muscle fibers, both syntrophin and β-dystroglycan are enriched in costameres (Williams, M.W., and R.J. Bloch, manuscript in preparation), as previously reported for dystrophin itself (see above). In mdx tissue, the amounts of β-dystroglycan and syntrophin decrease significantly, but low levels of both proteins can still be found at the sarcolemma ( 7 , 56 , 58 ). We used mAbs against β-dystroglycan or syntrophin together with 9050 anti–β-spectrin to label longitudinal sections of mdx muscle. Both syntrophin and β-dystroglycan were enriched in costameric structures at the sarcolemma that lacked longitudinal strands and elements overlying M lines, as well as in regions of the sarcolemma that were more severely altered . Comparison of β-spectrin with either β-dystroglycan or syntrophin demonstrated extensive colocalization . Thus, the reduced amounts of syntrophin and β-dystroglycan at the sarcolemma of mdx muscle codistributed in the membrane skeleton together with β-spectrin. Vinculin is a protein involved in the attachment of actin filaments to the membrane at focal adhesions ( 28 ) that is also found in costameres in skeletal muscle fibers ( 59 , 69 ), where it codistributes with β-spectrin and dystrophin ( 62 ). We labeled mdx tissue with anti-vinculin mAbs and with 9050 anti–β-spectrin. We found that the distribution of vinculin was also altered at the sarcolemma of the mdx mouse. Like the proteins studied above, vinculin was much less regularly organized in mdx muscle than in control muscle, with gaps appearing because of the loss of longitudinal domains or domains overlying Z or M lines. Vinculin continued to colocalize with β-spectrin in these altered structures . This result contradicts a recent report ( 51 ) stating that the organization of dystrophin, but not vinculin, was altered at the sarcolemma of muscle fibers from patients with Becker muscular dystrophy. We also examined the distribution of utrophin expressed in mdx muscle ( 42 , 46 , 48 , 86 ). Recent results with muscles that lack both dystrophin and utrophin ( 16 , 30 ) support the idea that the expression of low levels of utrophin in mdx muscle may protect it from the severe damage seen in Duchenne patients ( 79 ). Labeling of mdx muscle by anti-utrophin antibodies was apparent over wide areas of the sarcolemma in the same kinds of altered arrays as described above. When we compared the distribution of utrophin to that of β-spectrin by double label immunofluorescence, utrophin codistributed with β-spectrin at the sarcolemma . Control experiments ensured that our observations were not due to cross-reaction of the species-specific secondary antibodies with inappropriate primary antibodies, or to bleedthrough of the fluorescent label from one channel into another . Antibodies to β-spectrin generated in chickens and used to label mdx and wild-type muscle, together with appropriate controls, gave the same results. Thus, it is unlikely that the coincident labeling for β-spectrin and the other membrane skeletal proteins at the sarcolemma of wild-type or mdx muscle fibers was artifactual. Therefore, our results suggest that several proteins that codistribute with β-spectrin in costameres at the sarcolemma of healthy muscle also codistribute with β-spectrin in the irregularly organized membrane skeleton of mdx myofibers. The changes we observed in the organization of the sarcolemma might simply reflect extensive changes in the cytoarchitecture of dystrophic myofibers ( 52 ). To assess the effects of the mdx phenotype on the organization of intracellular structures lying near the dystrophic sarcolemma, we used antibodies to α-actinin and to two integral membrane proteins of intracellular membranes, coupled with confocal laser scanning microscopy. α-Actinin is the main component of the Z disc and may be involved in anchoring the connections between the costamere and the contractile apparatus near the Z line (for review see 71, 76). Double labeling with mouse mAbs to the sarcomeric form of α-actinin and rabbit antibodies against β-spectrin showed α-actinin distributed normally at Z lines , even at sites near the sarcolemma that showed highly disrupted patterns of labeling for β-spectrin . Thus α-actinin does not become disorganized in mdx muscle, even at Z lines that are in close proximity to areas of the dystrophic sarcolemma with abnormal membrane skeletal arrays. The Ca-ATPase of the sarcoplasmic reticulum (SERCA) and DHPR of the transverse tubules are also readily studied in double label immunofluorescence protocols. SERCA is normally distributed in tubular elements surrounding the Z line and M lines, as well as in elements aligned with the longitudinal axis of the myofibers . DHPR is easily visualized in mammalian muscle as a double line at the junctions of the A and I bands, where the transverse tubules surround each sarcomere, including those near the sarcolemma (38; for review see 25). The distributions of both these membrane proteins showed no evidence of an altered organization in mdx fibers, even when they were examined in the same confocal plane as the dystrophic sarcolemma, visualized with 9050 anti–β-spectrin . These results suggest that the changes in the organization of muscle fibers in adult mdx mice are limited to the sarcolemma and closely associated proteins. This is consistent with ultrastructural studies that reveal little change in the sarcoplasm of adult mdx muscle ( 13 , 80 ). We used immunofluorescence labeling and confocal microscopy to examine the distribution of membrane skeletal proteins in healthy and dystrophic ( mdx ) mouse muscle. Our experiments were designed to test the hypothesis that the absence of dystrophin is accompanied by extensive changes in the organization of the sarcolemma. Our results strongly support this hypothesis by demonstrating widespread and potentially significant changes in the distribution of several peripheral and integral membrane proteins at the sarcolemma. Surprisingly, all the proteins we examined continued to codistribute at the altered sarcolemma despite the absence of dystrophin. Earlier studies from this laboratory indicated the presence of β-spectrin in transverse structures overlying the Z lines and M lines, and in longitudinal strands ( 62 ). We noted that the β-spectrin pattern in muscle from mdx mice and from patients with Duchenne muscular dystrophy “always appeared less ordered than in controls” ( 62 ). Here we confirm and extend these earlier observations and show further that the extent of damage sustained by dystrophic myofibers can vary widely. This variability probably occurs because of differences in contractile activity and the load experienced in the period before sampling ( 41 , 45 , 68 , 74 ), and previous cycles of degeneration and regeneration (14, 19, 53; for review see 22). Therefore, sampling a large number of myofibers may be necessary to reach any conclusions about the extent of spectrin-based membrane skeleton reorganization in dystrophic muscle. Although much of our research has focused on β-spectrin, many other membrane-associated proteins codistribute with β-spectrin in irregular cytoskeletal arrays found at the dystrophic sarcolemma. These include all of the following: Na,K-ATPase, present in a complex with spectrin in skeletal muscle (Williams, M.W., and R.J. Bloch, manuscript in preparation); utrophin, the dystrophin homologue; two dystrophin-associated proteins, syntrophin and β-dystroglycan (for review see 9, 55); and vinculin, a protein normally found at focal adhesions and other plasmalemmal sites associated with organized bundles of actin microfilaments (for review see 11). The fact that all of these proteins are enriched in costameres of dystrophic and healthy skeletal muscle suggests that their organization is coordinated (however, see 51). The identity of the protein (or proteins) responsible for coordinating the distribution of proteins at the sarcolemma is still unclear. The most likely candidate is actin because it binds to dystrophin, β-spectrin, and vinculin, but the evidence that actin is enriched at costameres is minimal ( 12 ). Furthermore, mutations in dystrophin that eliminate its actin-binding activity produce mild phenotypes (9, 10, 84; however, see 3). If the extent of disorganization of the membrane-associated cytoskeleton is related to the severity of dystrophinopathy (see below), then the mild effects of these mutations would argue against a primary role for actin in coordinating the organization of dystrophin and its associated proteins with other membrane skeletal elements at the sarcolemma. In contrast, loss of dystrophin's binding site for dystroglycan results in the severest dystrophinopathies ( 9 , 65 ). This suggests that dystroglycan or other ligands (e.g., γ-sarcoglycan; ref. 31) interacting with the COOH-terminal region of dystrophin may be the primary organizers. For example, syntrophin binds to the voltage-gated sodium channel ( 27 ) that also associates with spectrin through ankyrin ( 72 ), suggesting that it may link the spectrin-based membrane skeleton to dystrophin in healthy muscle. However, as deletion of the syntrophin binding site in the COOH-terminal region of dystrophin ( 2 ) is not associated with myopathy in mice ( 65 ), syntrophin–dystrophin interactions are probably not required to maintain the integrity of the sarcolemma. Utrophin, expressed in mdx muscle ( 42 , 46 , 48 , 86 ), may partially substitute for dystrophin at the sarcolemma ( 79 ). Although it concentrates at the sarcolemma and binds to many of the same ligands as dystrophin, utrophin is unlikely to organize sarcolemmal spectrin and vinculin. Utrophin is present in mdx muscle at levels significantly lower than the levels of dystrophin in wild-type muscle, but levels of spectrin and vinculin are not appreciably reduced in mdx muscle (our unpublished observations). Thus, if utrophin does help to link the dystrophin-associated cytoskeleton to spectrin and vinculin at the sarcolemma, the stoichiometry of this linkage must differ significantly from that present in normal muscle. Furthermore, although utrophin, dystrophin, and spectrin have been shown to codistribute in a membrane skeletal network in cultures of rat myotubes, this network does not contain vinculin (18, 64; Bloch et al., manuscript in preparation). These results suggest that utrophin is unlikely to coordinate the organization of the various membrane skeletal proteins in mdx muscle fibers. Although the proteins that organize the membrane skeleton in dystrophic muscle must still be identified, dystrophin clearly plays an important role in maintaining the organization of the membrane skeleton in normal muscle. In dystrophinopathies, several of the structural domains at the sarcolemma of wild-type muscle tend to disappear. However, the nature of the relationship between this reorganization of the membrane skeleton and the absence of dystrophin in mdx muscle fibers is still not clear. The reorganization of the membrane skeleton in mdx myofibers may be an indirect result of the absence of dystrophin, perhaps related to changes in Ca 2+ homeostasis ( 21 , 33 , 36 , 44 , 66 , 78 ). Ca 2+ -dependent proteases, such as calpain and caspases, activated by Ca 2+ entering the myofiber through the dystrophic sarcolemma, are capable of cleaving spectrins ( 34 , 61 , 67 , 81 ) and other structural proteins ( 29 ). The proteolysis of spectrin, coupled with the absence of dystrophin, might disrupt the membrane-associated cytoskeleton sufficiently to destabilize sarcolemmal organization. This model predicts an increase in mdx muscle of proteolytic fragments of spectrin, perhaps associated with the altered costameric structures described here. Alternatively, the changes in sarcolemmal organization may not be directly linked to the absence of dystrophin at all. For example, they may be related to the stage of muscle regeneration reached by each dystrophic myofiber at the time of sampling. The polygonal arrays seen in a small percentage of mdx fibers also appear in myofibers developing in vivo (our unpublished results). However, myofibers in the mdx mouse tend to undergo only a single cycle of degeneration and regeneration occurring 3–5 wk after birth. Afterwards, only a small number of muscle fibers degenerate at any given time ( 13 , 14 , 53 ). As our current studies were limited to 6-mo-old animals, it is unlikely that this alone can account for our observation that the membrane skeleton is significantly altered in >90% of mdx muscle fibers . Our results may also be interpreted in terms of models that invoke a direct role for dystrophin in organizing the membrane skeleton in healthy muscle fibers. For example, dystrophin in healthy muscle may stabilize the costameric sites at which membrane skeletal proteins accumulate. Therefore, the absence of dystrophin in mdx muscles may render their membrane skeleton more susceptible to distortion and disruption by the forces exerted on the sarcolemma during muscle contraction and relaxation. This suggests that the membrane domains most easily disrupted at the sarcolemma are the longitudinal strands and the structures lying over M lines. These are more readily lost in mdx muscle, whereas sarcolemmal structures located over Z lines are more stable. Our previous studies of fast twitch rat muscle fibers have shown that the domains over Z lines contain β-spectrin complexed with α-fodrin, whereas the longitudinal strands and domains over M lines contain β-spectrin with little α-fodrin or any other α subunit of the spectrin superfamily that we have been able to identify ( 63 ). Such differences in the structure of membrane-bound spectrin may account for the greater instability of longitudinal and M line domains in mdx muscle. Structural differences may also explain why normal contractions lead to damage and degeneration of dystrophic muscle fibers. The altered organization of costameres suggests that the connections between the sarcolemma and the sarcomeres of superficial myofibrils are not as periodic in mdx muscle as they are in controls. In healthy myofibers, these connections mediate the transmission of force from the contractile apparatus to the sarcolemma and the extracellular matrix, while keeping the sarcolemma in close register with the underlying myoplasm during the contractile cycle ( 75 ). The reduced number and the abnormal arrangement of such connections are likely to render the dystrophic sarcolemma more fragile than controls. Disruption of the membrane skeleton of mdx also generates regions of the sarcolemma that are devoid of costameres. These regions can cover relatively large areas . In normal fast twitch muscle fibers of the rat, where longitudinal strands and structures overlying M lines are normally present, the regions between costameres usually do not exceed 2 μm 2 in area, and even these small regions are likely to be stabilized by low levels of dystrophin (Williams, M.W., and R.J. Bloch, manuscript in preparation). This protein is, of course, absent in mdx muscle. The gaps in the membrane skeleton of dystrophic myofibers are considerably smaller than the “delta lesions” documented in samples of Duchenne muscle ( 52 ), and unlike delta lesions they appear to be limited to structures at or immediately adjacent to the sarcolemma. Nevertheless, areas lacking a membrane skeleton may be especially susceptible to damage; indeed, they may even be the sites at which lesions are initiated. The reorganization of the sarcolemma and the appearance of gaps in the membrane skeleton may therefore lead directly to the damage sustained by dystrophic muscle fibers. It follows that measuring the reduction in the number and size of these gaps may provide a sensitive and quantitative way to test therapies now being developed to treat Duchenne muscular dystrophy. | Study | biomedical | en | 0.999995 |
10087269 | To determine whether properties of the hydrocarbon tail could efficiently target lipids after internalization, we used the dialkylindocarbocyanine (DiI) 1 series of lipid analogues. These analogues have varying propensities to partition into coexisting lateral membrane domains of varying fluidity and have different relative head group to tail cross-sectional areas resulting in varying overall shapes (approximated as cone, cylinder, or inverted cone). The DiI analogues are composed of an indocarbocyanine head group and two hydrophobic alkyl chains , which impart to them an overall amphiphilic character and allow them to insert into the membrane with their head groups roughly normal to the plane of the bilayer in a manner analogous to naturally occurring lipids. The relatively long hydrophobic alkyl chains result in their strong association with the host plasma membrane such that once inserted, they traffic as an integral part of it. Since the DiI derivatives are not naturally occurring lipids, they are not subject to intracellular metabolic turnover. As shown in Fig. 1 , two of the analogues we used contain saturated alkyl chains. DiIC 16 (3) (1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) has tails with 16 carbons each, whereas DiIC 12 (3) (1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) has 12 carbon chains. The differential partitioning preferences of C n DiIs into domains of varying motional characteristics have been investigated in model membrane systems. In systems with coexisting gel and fluid phases, an approximate match of the probe alkyl chain length with those of the host lipid acyl chains led to a preferential partitioning of the probe into gel phases . The alkyl chain length of DiIC 16 (3) approximately matches those most prevalent in the lipids of various CHO cell lines . Thus, in CHO cell membranes, DiIC 16 (3) would be expected to preferentially partition into more rigid (or highly ordered) domains, whereas DiIC 12 (3) would enter more fluid domains. The other lipid analogue used in this study, FAST DiI (1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), has two 18-carbon chains with two cis double bonds in each chain. Lipids with unsaturated tails preferentially enter fluid domains in model membranes containing coexisting gel and fluid phases . In rat basophil leukemia (RBL) cells, preferential segregation of DiIC 16 (3) into specific lateral domains containing aggregated immunoglobulin E receptors has been observed . Furthermore, during phagocytosis of 6-μm beads, RBL cells specifically exclude DiIC 16 (3) but not FAST DiI from the forming phagosomes . In addition, the overall shapes of the different DiI derivatives used in this study are quite different, which, in turn, would mean that they would have differential partitioning preferences into membrane regions of varying curvatures . Both DiIC 16 (3) and DiIC 12 (3) have a head group cross-sectional area that is larger than the alkyl chains (provided the chains are stretched out all-trans). They would thus exhibit an inverted cone shape and preferentially partition into membrane regions with a convex curvature. FAST DiI has a cylindrical or cone shape by virtue of the cis double bonds and would be preferentially accommodated in membranes of concave curvature. Thus, the set of DiI probes chosen for the present study are ideally suited to address the role of membrane domains in intracellular sorting since they vary in both their fluidity and curvature preferences. As seen in Fig. 1 , the DiO derivatives are identical to their DiI counterparts except that the head groups of DiO contain an oxygen atom as part of the heterocyclic ring system, instead of a carbon atom attached to two methyl groups in DiI . It is thus likely that the DiI and DiO head groups would interact differently with neighboring lipids and/or proteins. Thus, if both DiI and DiO derivatives with the same alkyl chain chemistry traffic identically in CHO cells, it would reinforce the argument that the alkyl chain properties are important in trafficking. To test a larger variation in head groups, we used phosphatidylcholine derivatives that have a zwitterionic head group, unlike the anionic DiI and DiO head groups . One long 16-carbon acyl chain ensured stable incorporation into the membrane bilayer. We used BODIPY FL lipid analogues since the fluorophore has been reported to localize to the membrane interior in a manner roughly normal to the plane of the bilayer , whereas in NBD lipid analogues the fluorophore loops back toward the hydrophilic interface . Furthermore, given the size of the BODIPY FL fluorophore, it is expected that incorporating it at the end of a 12-carbon acyl chain would result in a derivatized acyl chain that would roughly span the thickness of one membrane leaflet in a CHO cell. BODIPY FL C 12 -HPC [2- (BODIPY - 3 - dodecanoyl) - 1 - hexadecanoyl - sn - glycero-3-phosphocholine] would thus be somewhat similar to the DiIC 16 (3) or DiOC 16 (3) in terms of the properties of the hydrophobic tail. BODIPY FL C 5 -HPC [2-(BODIPY-3- pentanoyl) - 1 - hexadecanoyl- sn -glycero- 3- phosphocholine] would, on the other hand, be expected to more closely mimic DiIC 12 (3). All fluorescence probes were obtained from Molecular Probes Inc. The purity of the lipid analogues was checked by thin layer chromatography using chloroform/methanol/water (65:35:5 vol/vol) as the solvent system. Labeled dextran was dissolved in PBS, pH ∼7.4, and was extensively dialyzed before use to remove any unconjugated dye. Cy3 was obtained as a protein conjugation kit from Amersham Life Sciences . Human transferrin (Tf) was obtained from Sigma Chemical Co. It was then iron loaded and passed through a Sephacryl S-300 gel filtration system as previously described . Succinimidyl ester of Oregon green, Alexa 488, and Cy3 were then separately conjugated to the iron-loaded Tf following the manufacturer's instructions. Labeled transferrin was dialyzed thoroughly to remove the unbound dye. DiIC 18 (3)- labeled low density lipoprotein (DiI-LDL) was a gift from Dr. R.N. Ghosh (Cornell University Medical College, NY). All tissue culture supplies were from GIBCO BRL . All other chemicals were from Sigma Chemical Co. CHO cell lines expressing the human Tf receptor were grown in bicarbonate-buffered Ham's F-12 medium supplemented with 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 200 μg/ml geneticin. Geneticin was used as a selection for the transfected Tf receptors. All cells were grown in a 5% CO 2 environment in humidified incubators set at 37°C. The cells for microscopy were grown on 35-mm plastic tissue culture dishes whose bottoms were replaced with poly- d -lysine–coated coverslips, as described previously . All experimental manipulations as well as microscopy were carried out in these dishes. Stock solutions of the DiI derivatives were made in ethanol and stored at −86°C under argon. For making the labeling solutions, 750 nmol of DiIC 16 (3) or 75 nmol of DiIC 12 (3) or FAST DiI were dissolved in 400 μl ethanol. The ethanolic solutions were then injected, while vortexing, into an equimolar amount of fatty acid–depleted BSA in 1 ml PBS at pH 7.4 (final ethanol concentration 40% vol/vol). This mixture was then dialyzed thoroughly against several changes of PBS. As the ethanol was slowly exchanged during dialysis, some of the DiI got transferred to the hydrophobic fatty acid binding sites on the BSA, while much of it self-aggregated, and remained suspended in the labeling solution. The dialysate was then centrifuged twice at 100,000 g for 20 min each. When the supernatant from this procedure was run on a Sephacryl S-300 gel filtration column, the DiI eluted as a single peak associated with the BSA. Absorption spectrophotometric analysis showed a loading efficiency (DiI/BSA; mol/mol) of ∼0.1 for DiIC 16 (3), 0.3 for DiIC 12 (3), and 0.25 for FAST DiI. The DiI-loaded BSA solutions were sterilized by passage through 0.2-μm syringe filters and stored at 4°C under argon. Cells on coverslip-bottom dishes were taken out of the CO 2 incubator, rinsed several times with isotonic Medium 1 (150 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 20 mM Hepes, pH 7.4; supplemented with 2 g/liter glucose), and then labeled with an appropriate dilution of a DiI labeling solution (2 μM DiIC 16 (3), 31 nM DiIC 12 (3), and 75 nM FAST DiI). The concentrations of different DiI derivatives were optimized such that the final concentration of all analogues in the cells (as assessed by integrated fluorescence power per cell) were roughly matched. We used the minimum concentration in each case that would give us a useful fluorescence signal. All labeling reagents were ultracentrifuged at 100,000 g for 20 min just before an experiment and equilibrated to 37°C. When cells were labeled using these labeling solutions at 0°C, we obtained very good labeling of the plasma membrane. However, there was also extensive labeling of the background (predominantly the extracellular matrix). This background was dramatically reduced when labeling was instead carried out for a very short time (2 min) at 37°C. Thus, the latter approach was used for all the experiments presented here. After labeling, the cells were rinsed with ice-cold Medium 1 and fixed lightly with 2% paraformaldehyde for 10 min at 0°C. Fatty-acid free BSA was loaded with the other lipid analogues used in this study using identical procedures. Final concentrations of the different fluorophores used to label cells were 2 μM DiOC 16 (3), 150 nM FAST DiO, 30 nM BODIPY FL C 5 -HPC, and 1 μM BODIPY FL C 12 -HPC. Endocytic fates of the DiI derivatives were determined by comparing their intracellular distributions with those of the endocytosed Tf at various time points after the initial loading of the DiI derivatives on to the plasma membranes of TRVb-1 cells. Transferrin, bound to its receptor, was used as a marker for the endocytic recycling route . A comparison of the trafficking of various DiI derivatives with Tf thus allowed us to determine the degree of overlap of the endocytic trafficking routes of these DiI derivatives with that of Tf. The general experimental methods used to analyze the endocytic behavior of various DiI derivatives were as follows. Cells equilibrated to 37°C were labeled for 2 min at 37°C with the appropriate dilution of a DiI labeling solution, rinsed several times with Medium 1, and then incubated with pre-warmed Oregon green–labeled Tf (OG-Tf; or Alexa 488–conjugated Tf in the confocal experiments) for either 5 or 30 min. At the end of the incubation period, the cells were rinsed with ice-cold Medium 1 and fixed. To confirm the identity of the punctate structures labeled by some DiI derivatives as late endosomes/lysosomes, TRVb-1 cells were labeled with different DiI derivatives as described above, rinsed, and further incubated at 37°C with 1 mg/ml fluorescein-labeled dextrans for 60 min. After the incubation, the cells were rinsed and fixed as described above. Fluorescence microscopy and digital image acquisition were carried out using a Leica DMIRB microscope (Leica Mikroscopie und Systeme GmbH) equipped with a cooled CCD camera (Frame Transfer Pentamax camera with a 512 × 512 back-thinned EEV chip, No. 512EFTB; Princeton Instruments ) driven by Image-1/MetaMorph Imaging System software ( Universal Imaging Corp. ). All images were acquired using a high magnification (63×, 1.4 NA) oil immersion objective. DiI derivatives were imaged using a standard rhodamine filter set, while Oregon green was imaged using a fluorescein filter set. The fields to be imaged were chosen on the basis of well spread out cell morphology and the focal plane was chosen to have the structure of interest (e.g., the endocytic recycling compartment [ERC]) in focus in the green (OG-Tf) channel. Choosing the areas for imaging in the non-DiI channel was especially important for the quantitative analyses, since the labeling pattern of the DiI derivatives varied significantly from cell to cell, and this distribution could be potentially skewed by observer bias if the focal plane was chosen in the DiI-labeled fields. We chose Oregon green over fluorescein to label Tf for quantitative microscopy since Oregon green has spectroscopic properties similar to fluorescein, but it has significantly higher photostability and a lower pKa and higher fluorescence yield than fluorescein . All image analysis was carried out using the Image-1/MetaMorph Imaging System software. For quantitative analyses, the images of cells double labeled with OG-Tf (green) and one of the DiI derivatives (red) and a corresponding differential interference contrast image were sequentially acquired using the CCD camera (12 bit format) and were processed as follows. The fluorescence images were first background corrected by applying a median filter using a 64 × 64 pixel area (0.24 μm/pixel), and the background image was subtracted from the acquired image. The degree of crossover of signal from one channel to the other was determined using cells labeled singly with each of the fluorophores. These images of singly labeled cells were background corrected, followed by measurement of integrated fluorescence intensity in the whole field in both channels. Since autofluorescence was negligible at the exposures used for these experiments, the fluorescence intensity observed in the unlabeled channel (after background subtraction) was taken to represent signal crossover. Crossover measurements were made for four different fields for each fluorophore and averaged to obtain a representative crossover fraction. This crossover fraction of each image was then subtracted from the corresponding image in a double-labeled set before further analysis. The crossover intensity was <2% of the true fluorescence intensity in all cases. The cell outlines for each set of double-labeled fields were traced out manually in the corresponding differential interference contrast image, and then copied on to the Tf- and DiI-labeled fields. In each cell, the whole cell green (Tf) and red (DiI) fluorescence intensities were measured. Simultaneously in each cell, the relative intensity in the ERC was measured by placing a small box (4 × 4 pixel area) in several locations within the morphologically defined perinuclear recycling compartment. In case of the DiI-labeled fields, precaution was taken not to include any punctate structures that may lie within the rather large area occupied by the ERC. The ratio of fluorescence intensities sampled within the ERCs for the DiI derivative to Tf was then normalized by the whole cell fluorescence intensity ratio for that cell to correct for cell to cell variation in Tf receptor expression and DiI uptake. For the results presented in Figs. 7 – 9 , the measurements were made for ∼50 cells in each data set, and the data were collected from experiments carried out on two different days. The validity as well as the limits of this method were first tested using control experiments (see Results). Confocal microscopy was performed using an Axiovert 100M inverted microscope equipped with an LSM 510 laser scanning unit and a 63× 1.4 NA plan Apochromat objective (all from Carl Zeiss, Inc. ). Samples were excited with a 25-mW argon laser emitting at 458 and 488 nm and a 0.5 mW helium/argon laser emitting at 543 nm; emissions were collected using a 505–530-nm band pass filter to collect green (Alexa 488) emission and a 585-nm long-pass filter to collect red (DiI) emission. Confocal slices were obtained in 0.2-μm increments. The images were collected in eight-bit format, exported from the LSM 510 software, and each confocal slice was background corrected (using a 64 × 64 pixel median filter) using the MetaMorph software. Summation projection of all background corrected confocal slices were also produced using the MetaMorph software. For visual output purposes, the digital images (both wide-field and confocal) were clipped to the relevant eight bits, transferred to a Macintosh Power PC, and intensity mapped through logarithmic look up tables (luts) using Adobe Photoshop software and printed on a dye sublimation printer (SpectraStar Dsx; General Parametrics Corp.). For quantitative analyses, the data obtained from MetaMorph Imaging System were routinely transferred to Microsoft Excel spreadsheet software for further calculations. For our studies, we needed to develop a method of incorporation of different DiI derivatives in the plasma membranes of TRVb-1 cells that would not produce particulates containing DiI. Such particulates could potentially produce artifacts in our experiments, since they could either be pinocytosed by cells or stick nonspecifically to the cell surface and slowly diffuse from there. We found that many previously published labeling protocols produced high levels of aggregated DiI in the aqueous loading buffers. It was difficult to remove these particles by centrifugation since most of these protocols required the use of organic solvents, and at the high speeds necessary to remove all the particles there was a separation of the organic phase with most of the DiI probes dissolved in it. As described in Materials and Methods, we developed a labeling protocol in which DiI derivatives were transferred from an ethanolic stock solution to fatty acid–free BSA in aqueous solution. Ethanol was then removed by dialysis, and the particulates were removed by ultracentrifugation. This method produced a labeling solution with no DiI particles detectable by fluorescence microscopy or gel filtration chromatography. Fig. 2 shows TRVb-1 cells labeled using these DiI/BSA labeling solutions after appropriate dilution at 37°C for 2 min. All three DiI derivatives exhibited plasma membrane labeling with no gross heterogeneity in distribution. There is some variation in intensity that is consistent with surface projections or a small amount of endocytosis during the 2-min incubation. To ensure that the DiI labeling the cell surface had been released from albumin, we did a control experiment in which cells were incubated with a labeling solution containing BSA covalently conjugated to fluorescein (data not shown). No detectable fluorescein signal was left after the labeled BSA was rinsed away. Since the DiI derivatives used in this study label the plasma membrane uniformly, they would be expected, a priori, to enter the cells through all available endocytic routes. While these pathways appear to merge at the level of the peripherally distributed tubulovesicular sorting endosomes in most cell types , in some cases, such as ruffling A431 cells, they have been reported to remain segregated from each other . It was thus important to document that all the DiI analogues used in this study predominantly entered sorting endosomes, which were characterized by the presence of fluorescent Tf at early times after the initiation of endocytosis of the fluorescent markers. We achieved this by double-labeling TRVb-1 cells for very short times (1 min) with a mixture of 15 μg/ml OG-Tf and each of the DiI derivatives, followed by immediate fixation and looking for colocalization of OG-Tf and the DiI analogues . Although only a small fraction of the DiI was internalized in 1 min, most vesicles with detectable DiI contain Tf. In some Tf-labeled vesicles, it is difficult to see the DiI because of the high plasma membrane background. We conclude that both Tf and the DiI derivatives enter the same sorting endosomes. Intracellular fates of the DiI derivatives were followed by comparing their trafficking to that of receptor-bound OG-Tf, which follows the endocytic recycling pathway. In TRVb-1 cells, Tf exits the sorting endosome with a t 1/2 of ∼2 min and is delivered to the ERC . Exit from the ERC and delivery to the cell surface occurs with a t 1/2 of 10–12 min . In TRVb-1 and other CHO cell lines, the ERC is a collection of narrow tubular elements that organize near the microtubule organizing center, and when labeled with a fluorescent marker the ERC appears as a large perinuclear fluorescent spot . Since exit from the ERC is the slowest step in the endocytic recycling itinerary of the Tf receptor, the ERC is the most brightly labeled structure at steady state . The following experiments were designed so that the cell surface Tf receptors would always be saturated with Tf, and the internalization of plasma membrane–associated DiI derivatives could be compared with Tf. Fig. 4 shows cells that were labeled with 2 μM DiIC 16 (3) for 2 min, washed, and then incubated with 10 μg/ml OG-Tf for 5 (a and b) or 30 (c and d) min. We observe that after 5 min of internalization, OG-Tf is mainly in the ERC, which appears as a single area of fluorescence near the center of each cell . In contrast, a significant fraction of the DiIC 16 (3) appears in punctate, vesicular structures . A small fraction of DiIC 16 (3) does appear to codistribute with the Tf at early times. By 30 min, nearly all internalized DiIC 16 (3) is found to segregate away from Tf, and appears in discrete punctate structures that are distributed throughout the cell . In similar experiments (data not shown), we find that even at earlier times after endocytosis (1–5 min) there is never a significant concentration of DiIC 16 (3) in the pericentriolar area. Most of the DiIC 16 (3) remains in punctate structures that remain discrete and increase in brightness over this period. This suggests that most DiIC 16 (3) is retained in the sorting endosome and does not exit along with transferrin receptors that are delivered to the endocytic recycling compartment. To examine whether the small fraction of DiIC 16 (3) that appears to colocalize with OG-Tf at 5 min by wide-field microscopy is indeed in the same morphological structures as Tf, we carried out confocal microscopy of cells double labeled with DiIC 16 (3) and Alexa 488–labeled Tf after a 5-min chase. Fig. 4 , e and f, shows a single optical section through these cells, while Fig. 4 , g and h, shows a summation projection through all the optical sections through the cell. As is clear from Fig. 4 , e and f, a fraction of DiIC 16 (3) at this time does localize to structures that overlap with Tf-containing ERC and cannot be accounted for by out-of-focus fluorescence from late endosomes/lysosomes containing DiIC 16 (3). However, the summation projection images show that this population represents a small fraction of total intracellular DiIC 16 (3) fluorescence. Fig. 5 shows the results from a similar experiment in which the cells were double labeled with OG-Tf (a and c) and DiIC 12 (3) (b and d), and Fig. 6 shows cells that were double labeled with OG-Tf (a and c) and FAST DiI (b and d). Figs. 5 and 6 show the relative distributions of OG-Tf and the DiI derivative after 5 (a and b) and 30 (c and d) min of endocytosis. The DiI derivatives seem to traffic in a fashion qualitatively similar to Tf (a and c), at least until 30 min after initiation of endocytosis. This was in contrast to the trafficking of DiIC 16 (3), where after 30 min of endocytosis nearly all DiIC 16 (3) was found in punctate structures devoid of Tf. To better understand the trafficking of the DiI derivatives in relatively large cell populations, we carried out a quantitative analysis of the colocalization of various DiI derivatives and Tf in the ERC. The objective was to obtain an estimate of how similar the trafficking of each of the DiI derivatives was to that of Tf, without explicitly defining the alternate destinations of the DiI derivatives. Such an estimate could be obtained from the ratio of the fluorescence intensities of a DiI derivative to that of Tf in the region of the ERC after varying periods of endocytosis. In this analysis, a ratio closer to 1 indicates a trafficking behavior more similar to Tf. To account for differences among cells in the total amount of each fluorophore per cell, we normalized the ratio of fluorescence intensities in the ERC region of every cell to the ratio of the total fluorescence of the two fluorophores in that cell. The details of the image analysis protocol are discussed in Materials and Methods. We tested the validity of this image analysis protocol and determined the upper and lower limits on the ratios in the ERC that could be reliably measured. In the first test case, the cells were labeled with a mixture of OG-Tf (green fluorescence) and Cy3-Tf (red fluorescence) continuously for 30 min. Fig. 7 a shows a frequency histogram ( n = 50) comparing the distribution of the two Tfs. As expected for molecules that traffic identically, we obtained a narrow distribution of ratios in the ERC that is centered around 1. To test the case where two probes should sort efficiently from each other, we double-labeled cells with a mixture of DiI-LDL (which is trafficked to the late endosomes) and OG-Tf (which recycles efficiently). The cells were labeled for 5 min with a mixture of both probes at 37°C, followed by a 5-min chase. In this case , we obtained a relatively narrow distribution of ratios of LDL/Tf in the ERC region that was centered around 0.25. The nonzero value of this ratio is due to overlap of the fluorescence from DiI-LDL containing late endosomes/lysosomes that happen to localize in the region of the ERC labeled with OG-Tf. Thus, a test molecule that is completely sorted away from the recycling route would still be expected to show a nonzero ratio to Tf in the ERC region using this image analysis protocol. Fig. 8 shows a quantitative analysis of the ratios of various DiI derivatives to Tf in the ERC regions of TRVb-1 cells ( n = 50 for each data set). Fig. 8 , a and d, shows the distribution of the ratios in the ERC for DiIC 16 (3), b and e for DiIC 12 (3), and c and f for FAST DiI. Fig. 9 shows the mean ratio of each DiI derivative to Tf in the ERC at each time point for easier comparison. The analysis shows that even at early times (5 min of endocytosis), the distributions for the three DiI derivatives are significantly different from each other. While FAST DiI shows the highest degree of colocalization with Tf in the ERC (ratio centered around 0.8), DiIC 16 (3) shows the lowest ratio (centered around 0.4) with DiIC 12 (3) showing an intermediate behavior. This means that FAST DiI and DiIC 12 (3) traffic in a manner more like Tf, compared with DiIC 16 (3). These results show that sorting of DiIC 16 (3) occurs relatively quickly after endocytosis, with a significant difference in the ratio in the ERC by 5 min. By 30 min, the differences among various DiI derivatives becomes more pronounced. They all sort away from the Tf recycling pathway to different degrees, with the difference being most pronounced for DiIC 16 (3) . In fact, the distribution of the ratio values for DiIC 16 (3) to Tf is similar to the distribution for LDL to Tf . From the results presented above, we find that DiIC 16 (3) separates from Tf very efficiently following endocytosis into sorting endosomes, and by 30 min its overlap with Tf in the ERC region is similar to the overlap seen for LDL. DiIC 12 (3) and FAST DiI show greater overlap with Tf. However, in none of these cases does the ratio of a DiI derivative to Tf reach 1 (as seen when two differently tagged Tfs are used to label the cells). This indicates that the DiI derivatives exhibit a range of partitioning preferences that lead to differential trafficking, and the sorting is not all-or-none. We also note that the distributions of the ratios of various DiI derivatives to Tf in the ERCs of CHO cells are significantly wider than a similar distribution for the cells double labeled with Cy3-Tf/OG-Tf or LDL/Tf . This is indicative of cell-to-cell variability in the trafficking of the DiI derivatives. The basis for this heterogeneity is unknown. We used colocalization with high molecular weight fluorescein-labeled dextrans as an assay to test whether the punctate structures that contained the DiI derivatives at later times were late endosomes/lysosomes. In Fig. 10 , we show a colocalization of the various DiI derivatives with fluorescein dextran after a 60-min chase. The cells were labeled for 2 min at 37°C with each DiI derivative, washed, and then incubated for 60 min in the presence of 1 mg/ml fluorescein dextran. Fig. 10 , a, c, and e, shows the distribution of fluorescein dextran in cells double labeled with DiIC 16 (3) (b), DiIC 12 (3) (d), and FAST DiI (f). Fig. 10 , a and b, shows a substantial overlap between DiIC 16 (3) and fluorescein dextran after a 1-h chase, with a more modest fraction of either DiIC 12 (3) (c and d) or FAST DiI (e and f) entering these compartments. Fluorescence from the DiI derivatives in the ERC is not seen in Fig. 10 because the ERC is at a higher focal plane than the late endosomes in most cells. These results show that, among the DiI derivatives investigated in this paper, only DiIC 16 (3) enters late endosomes/lysosomes in significant proportions. To ensure that the endocytic sorting of the DiI derivatives reported in this paper is not unique to these fluorophores, we conducted similar experiments with two other sets of lipid analogues. One set, DiOC 16 (3) and FAST DiO, were identical to the DiI analogues except that they contained an oxygen atom instead of a carbon atom attached to two methyl groups in their head groups . Both DiO derivatives contained long chains, but in the former case they were both saturated, while in the latter they contained two double bonds each. The other set constituted of two analogues of phosphatidylcholine, which both had one saturated 16-carbon tail, whereas the second tail contained a BODIPY FL fluorophore at the end of either a 5- or a 12-carbon acyl chain . Fig. 11 shows the distribution of these lipid analogues in TRVb-1 cells singly labeled with each fluorophore. Cells were labeled with 150 nM FAST DiO , 2 μM DiOC 16 (3) , 30 nM BODIPY FL C 5 -HPC , and 1 μM BODIPY FL C 12 -HPC . For each type of lipid analogue, the concentrations of the labeling solutions were adjusted to provide approximately the same level of incorporation in the cells. The cells were labeled for 2 min at 37°C with each labeling solution, rinsed, incubated further at 37°C in Medium 1 for 30 min, and then fixed lightly with paraformaldehyde. The results show that while the lipid analogues with unsaturations in their tails or containing one short tail predominantly entered a central fluorescent compartment; lipid analogues containing long saturated tails (e.g., DiOC 16 (3); Fig. 11 b and BODIPY FL C 12 -HPC; Fig. 11 d) entered punctate structures distributed throughout the cells. Double-labeling studies (data not shown) confirm the identities of the central fluorescent compartment as the ERC and the punctate structures as late endosomes/lysosomes. We do observe some differences in the degrees to which DiOC 16 (3) and BODIPY FL C 12 -HPC are directed to the late endocytic pathway. While DiOC 16 (3) appears to be as efficient as DiIC 16 (3) in being delivered to the late endosomes in 30 min, we see a larger fraction of BODIPY FL C 12 -HPC in the ERC at this time. However, as seen in Fig. 11 , the difference between these analogues and the corresponding analogues with unsaturated and short chains is very clear. These results are similar to our observations with the DiI analogues in which lipid analogues with short or unsaturated tails recycle efficiently, but those with long and saturated tails are preferentially directed to the late endocytic pathway. The results presented here show that different DiI derivatives, varying solely in the composition of their alkyl chains, exhibit differential trafficking after their internalization from the cell surface into sorting endosomes. While the DiI derivative with long and saturated tails [i.e., DiIC 16 (3)] preferentially enters the late endocytic pathway, the derivatives with shorter [DiIC 12 (3)] or unsaturated ( FAST DiI) tails are efficiently delivered to the ERC. These observations are reinforced by two other sets of lipid analogues. There are several steps along the endocytic pathway where sorting of different DiI analogues could have occurred. First, some of the segregation might have occurred at the plasma membrane. Domains enriched in DiIC 16 (3) have been observed on the plasma membrane of rat basophil leukemia cells when IgE receptors are cross-linked . Although we did not detect any inhomogeneity in the distribution of the DiI derivatives on the cell surface, we cannot rule out submicroscopic segregation and/or variations in the rates of internalization of different DiI derivatives. However, even if cell surface heterogeneity is present, it could not account for the sorting we observe between DiIC 16 (3) and the other two DiI derivatives, since we report that all DiI derivatives initially enter sorting endosomes that also contain endocytosed Tf. Thus, most of the sorting must take place at a step after the internalization of DiIC 16 (3) and Tf into common sorting endosomes. The sorting endosomes serve as the branch point for entry into either the recycling route or the late endocytic route. We propose that this is the major site where DiIC 16 (3) sorts out of the endocytic recycling route. This site of sorting is consistent with the fact that the sorting occurs as early as 5 min after endocytosis. DiIC 16 (3) is retained in punctate endosomes while FAST DiI begins to enter the ERC within 5 min. As expected for a molecule that is retained in the sorting endosomes, nonrecycled DiIC 16 (3) is subsequently delivered to the late endosomes . The retention of DiIC 16 (3) in the sorting endosomes and its subsequent delivery to the late endocytic structures does not appear to be determined, in any significant way, by specific lipid–protein interaction with some transmembrane protein. In control experiments (data not shown), we have seen that DiIC 16 (3) is delivered to late endosomes even when the cells are labeled with 10-fold more DiIC 16 (3) than used in the experiments reported in this study. It has been estimated in the case of rat basophilic leukemia cells that optimal labeling of the cell surface with DiIC 16 (3) results in ∼10 7 –10 8 DiI molecules per cell . On the contrary, even a highly expressed protein on the cell surface has ∼10 5 –10 6 copies . Considering that such a protein would also interact with native lipids, it seems unlikely that stoichiometric binding to any transmembrane protein or even a group of proteins would redirect all DiIC 16 (3) to the late endosomes. In contrast, if lateral membrane domains of varying fluidity and/or curvatures did exist in the membranes of the sorting endosomes, DiIC 16 (3), by virtue of its long saturated alkyl chains, would partition preferentially into the more rigid domains or those with a convex curvature. We show several variations of these possibilities in Fig. 12 . We suggest that if any specific lipid–protein interaction has to occur, it would be feasible only within the context of fluidity- or curvature-dependent cosegregation of the lipids and proteins in question, so that high enough effective concentrations can be achieved. The differential trafficking of the lipid analogues with long saturated hydrophobic tails relative to those with short or unsaturated tails can be rationalized if the vesicular region of the sorting endosomes are considered to represent membrane domains that are more rigid than those in the attached tubules that bud from the sorting endosomes [see Fig. 12 , (1)]. There is evidence that narrow tubules in model membrane systems are enriched in more fluid domains. For example, when either gel or fluid phase liposomes were subjected to suction by a micropipette, it was found to be easier to pull fluid membranes into long narrow tubules in response to suction as compared with membranes in the gel phase . Further, a study that measured diffusion coefficients of transmembrane proteins in red blood cell “tethers” (hollow membranous cylinders, up to 36 μm long and ∼100 nm in diameter) found a dramatic increase in the diffusion coefficients of these proteins in the tether membrane . In an alternate model, the bilayer fluidity in both the vesicular and the tubular parts of the sorting endosome could be roughly similar, except that the necks joining the tubules to the vesicle, which are under high curvature stress, would be specifically enriched in lipids that can be accommodated in regions of high curvature [Fig. 12 , (2)]. In this case, any lipid analogue that cannot easily traverse this small but highly curved fluid (disordered) domain at the neck of the tubule would be preferentially retained in the sorting endosome. Both alternatives are feasible and have been proposed previously in biophysical treatments of bud formation in model membranes . In addition, the shape of an amphiphile can be related to its propensity to induce curvature in model membranes, as well as to partition preferentially into preexisting membrane domains of varying curvatures . More precisely, if a molecule has approximately equal cross-sectional areas in its head group and tail region, the molecule can be considered as a cylinder that does not induce any curvature in a bilayer and would preferentially partition into planar regions of the membrane (no curvature preference). On the other hand, amphiphiles with larger (an inverted cone) or smaller (a cone) head group cross-sectional areas would induce opposite curvatures or partition into preexisting regions of opposite curvature. Since DiIC 16 (3), with its alkyl tails stretched out all-trans, would approximate an inverted cone, it would be expected to preferentially enter intralumenal invaginations or involutions in the sorting endosome membrane and hence segregate from the recycling pool of membrane-bound molecules [Fig. 12 , (3)]. FAST DiI, on the other hand, would look more like a cylinder or a cone (depending on the number of gauche conformations in the alkyl tails in addition to the two cis double bonds) and hence would have either no curvature preference or a preference for concave curvatures that would result in its enhanced entry into the tubules of the sorting endosomes. This difference in shape might partly explain why DiIC 12 (3) recycles less efficiently than FAST DiI even though both have similar fluidity preferences. From the sorting endosomes, most membrane-bound components enter the ERC and are recycled out to the cell surface with a half time of 10–12 min. The ERC has been shown to have sorting functions of its own . Recent studies show that glycosylphosphatidylinositol (GPI)– anchored proteins are recycled approximately three times more slowly than C 6 -NBD-SM or Tf receptor . The recycling of GPI-anchored proteins can be restored to the same rate as Tf receptor when the cells are grown in media that lower cellular cholesterol by ∼40% . Cholesterol is known to be a major modulator of bilayer motional characteristics and to be involved in the formation of ordered domains in model membrane systems . The cholesterol dependence suggests the involvement of ordered lipid domains in the slow recycling of GPI-anchored proteins. The ERC does not, however, appear to be a major site for the sorting of DiIC 16 (3) from other recycling markers, since at no time during its endocytic itinerary do we see a significant concentration of DiIC 16 (3) in the ERC. As seen in Fig. 4 , there is a minor fraction of DiIC 16 (3) that appears to localize at the ERC by optical microscopy. Even though some DiIC 16 (3) appears to enter the ERC, its efficiency of sorting to late endosomes would be comparable with that found for ligands that bind to receptors. For example, ∼20% of all internalized α 2 -macroglobulin and ∼40% of insulin are recycled undegraded during each endocytic passage through the cell. Several previously reported examples of lipid sorting can be understood in terms of our working hypothesis based on lipid shapes and fluidity preferences. For example, C 6 -NBD-SM and related lipid analogues that recycle efficiently contain one very short (6-carbon) acyl chain to which the reporter group is attached. The polar NBD group would make this tail loop up to the membrane interface , so that the total cross-sectional area occupied by this molecule would be quite large. These factors would be expected to result in preferential partitioning of this lipid analogue to more fluid regions of the bilayer, and thus would be predicted by our model to predominantly enter the endocytic recycling pathway. On the other hand, some lipids that have been reported to be targeted to late endocytic compartments contain long, saturated acyl chains that would result in their preferential partitioning to the more rigid parts of the bilayer. In fact, Sandhoff and Klein have suggested that the retention of certain lipid components such as glycosphingolipids in the inner involutions of the endosomes may play a role in their eventual delivery to the degradative pathway. Our observations are also consistent with the recent observations of Chen et al. . This study used fibroblasts whose plasma membranes were labeled with a BODIPY FL–labeled lipid analogue that fluoresced green at low concentrations (monomer fluorescence) and red at high concentrations (excimer fluorescence). The plasma membranes for these experiments were labeled such that all the cell surface fluorescence was only monomer (green) fluorescence. Interestingly, when these cells were allowed to endocytose for as short as 7 s, it was found that, while some newly formed endosomes still fluoresced green, others in the same cell exhibited red (excimer) fluorescence. All plasma membrane fluorescence still remained green. These observations could either mean that the lipid analogue segregated into lateral domains more or less enriched in this analogue just before being endocytosed, or that all endosomes contain a roughly similar number of copies of the fluorescent lipid analogue, but that in a subset of those endosomes the analogue segregated into subdomains, thereby increasing their effective concentration and giving rise to excimer fluorescence. Both these possibilities would be consistent with our working hypothesis for lipid sorting in fibroblasts. Sorting of lipids occurs at many membrane trafficking steps. For example, the sorting of DiIC 16 (3) out of the recycling pathway occurs primarily at the sorting endosomes, while GPI-anchored proteins are delayed in their export from the ERC. Similarly, the TGN has been reported by several groups to be the primary site along the biosynthetic pathway for sorting of apically and basolaterally destined lipids . It is conceivable that lipid sorting occurs to some extent at every step where a vesicle or tubule buds from an organelle. We emphasize that the possibilities presented in our model are neither exhaustive nor mutually exclusive. On the contrary, they are presented solely as pointers toward the various possibilities that are conceivable. In general, the principles involved in this type of sorting include the chemistry of the individual membrane component and the composition of the host bilayer. The process we observe in endosomes may very well reflect a more ubiquitous mechanism in several intracellular sorting processes. This would offer an energetically inexpensive mechanism for a relatively efficient sorting of certain classes of molecules from others . In the cellular milieu, such mechanisms would act in concert with other, more specific, protein– protein or protein–lipid interactions that could modulate or “fine-tune” this basal level sorting. | Study | biomedical | en | 0.999998 |
10087270 | Human caveolin-1 cDNA was generated by reverse transcription and polymerase chain reaction, subcloned into TA cloning vector (Invitrogen), verified by nucleotide sequencing (Sequenase; United States Biochemical), digested with BamHI and Xhol, and then subcloned into the inducible expression vector pML1 in an inverted order. Cells were transfected by electroporation, clones selected in hygromycin, and levels of caveolin-1 assessed by immunoblotting after overnight culture with 3 μM CdCl 2 . Caveolin cDNA was also subcloned into the CMV promoter-driven expression vector, pCEP9 (Invitrogen), and clones selected by growth in G418. Transfected cells were then screened for protein expression via immunoblot with antibody specific for caveolin-1 (Transduction Labs). Cells (5 × 10 4 ) were seeded in fibronectin (5 μg/ml; Sigma Chemical Co. ) or vitronectin (1 μg/ml; Becton Dickinson ) coated 96-well tissue culture plates, incubated at 37°C for 1 h, and adherent cells quantified as described previously . Before the adhesion assay, antisense caveolin 293 or uPAR/293 cells were induced with 3 μM CdCl 2 at 37°C for 8 h. Fibronectin binding was assessed on cells seeded in polylysine-coated 96-well tissue culture plates, cultured overnight, induced with 3 μM CdCl 2 at 37°C for 8 h, and then washed with binding buffer (DMEM, 1 mg/ml BSA). Half of the cells were treated with β1 integrin activating antibody TS2/16 (3 μg/ml; Endogen ) at 4°C for 30 min, and then all cells were incubated with buffer containing 125 I-fibronectin (1, 5, and 10 nM) at 4°C for 1.5 h. After washing, cells were lysed in 0.2% SDS, 0.2% Triton X-100, 10% glycerol in PBS for 30 min, and radioactivity was measured. Nonspecific binding was determined by inclusion of 0.5 mM RGDS (American Peptide Co.) in the reaction mixture. Cells transfected with caveolin antisense constructs were induced with 3 μM CdCl 2 at 37°C for 8–18 h. Cells were then detached and incubated with PBS containing 0.1% BSA and primary antibodies to uPAR (R2) or integrins β1 (JB1A) , α5β1 (Chemicon), and αv (L230) (American Type Culture Collection) on ice for 30 min. After washing, cells were incubated with FITC-conjugated goat anti–mouse IgG ( Sigma Chemical Co. ) and analyzed on a flow cytometer (FACScan ® ; Becton Dickinson ). To assess the distribution of caveolin and β1 integrins, human saphenous vein smooth muscle cells (SMC) and 293 or uPAR/293 cells coexpressing caveolin were cultured overnight on glass coverslips in 10% FBS. After washing, cells were incubated for 30 min with JB1A and/or K20 (Immunotec) antibodies to β1 or isotype controls at 4°C and fixed for 20 min in 3.7% paraformaldehyde. Fixed cells were permeabilized and incubated with rabbit polyclonal antibody to caveolin (Transduction Labs) for 30 min at room temperature, then incubated with FITC- or Rhodamine red– conjugated secondary antibodies and coverslips mounted in Prolong (Molecular Probes). Fluorescence staining was analyzed by confocal laser attached to a Zeiss microscope (model Axiovert S100) using separate filters for each fluorochrome. Optical planes were imported into Adobe Photoshop and processed. To visualize integrin and caveolin clustering, SMC in suspension were incubated with FITC-K20 β1 antibodies without or with goat anti–mouse secondary antibodies for 1 h at 37°C, immobilized on 50 μg/ml polylysine-coated coverslips for 10 min, and then fixed and permeabilized as above. Cells were stained for caveolin and analyzed by confocal laser microscopy. Control or antisense caveolin clones were cultured for 8 h in serum-free DMEM containing 3 μM CdCl 2 on either fibronectin (5 μg/ml), vitronectin (1 μg/ml), or polylysine (50 μg/ml) and lysed on ice for 30 min in buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethyl-sulfonylfluoride, and leupeptin (10 μg/ml). After preclearing with protein A–agarose, lysates were incubated with antibodies to β1 integrin (JB1A) or cortactin (Upstate Biotechnology Inc.) at 4°C overnight. In some experiments, the lysates were made 10 μM in the caveolin-related peptide, cav-1 (DGIWKASFTTFTVTKYWFYR), or the control cav-x peptide (WGIDKAFFTTSTVTYKWFRY), in 0.25% DMSO . The cav-x peptide has an identical amino acid composition and 60% identical sequence to the cav-1 peptide. The immunoprecipitates were blotted with caveolin antibody (Transduction Labs), stripped, and reblotted for Src family kinases ( Santa Cruz Biotech ), cortactin, or 4G10 (Upstate Biotechnology Inc.). To analyze FAK and EGF receptor activation, clones were induced with CdCl 2 and plated on fibronectin- or polylysine-coated 100-mm dishes or their β1 integrins clustered with antibodies. Cells were lysed or stimulated with EGF (10 ng/ml; Upstate Biotechnology Inc.) for 5 min before lysing in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with protease inhibitors. Lysates were immunoblotted for ErbB-2, -3, and -4 ( Santa Cruz Biotech ) and phosphotyrosine (Upstate Biotechnology Inc.). β1 immunoprecipitates from m1–m5 clones were immunoblotted with uPAR antibody 399R (American Diagnostica). To measure total enzymatic activity of β1-associated Src kinases, induced cells were lysed in Triton X-100 buffer and immunoprecipitated with β1 antibody (JB1A). The immunocomplexes were suspended in 24 μl of reaction solution containing 50 mM Pipes, pH 7.0, 10 mM MnCl 2 , 5 μg of acid-denatured enolase, 10 μM ATP, 10 μCi of [γ- 32 P]ATP, and reacted at room temperature for 10 min. The kinase reaction was stopped by sample buffer, and samples were resolved on 10% SDS-PAGE and visualized by autoradiography. For determination of basal activity of Src, induced cells were lysed in RIPA buffer and ∼500 μg of lysate immunoprecipitated with Src mAb ( Calbiochem ) and procedures described above followed. SMC were plated onto fibronectin- (5 μg/ml) or polylysine-coated (50 μg/ ml) dishes at 37°C for 20 min in serum-free DMEM/BSA with or without peptide 25 or 36 (75 μM). Cells were lysed in RIPA buffer and immunoblotted with phospho-specific MAPK antibody which detects p42 and p44 MAPK catalytically activated by phosphorylation at Tyr204/Thr202 ( New England Biolabs ). The membrane was stripped and reprobed for total MAPK protein as control. Passage 1–3 SMC (10 5 ) were seeded into Transwell (Costar; Becton Dickinson ) inserts containing 8-μm polycarbonate filters precoated on the bottom with fibronectin, and then cultured overnight in serum-free DMEM containing BSA (5 mg/ml) with or without peptide 25 or 36 (50–100 μM). Cells on both sides of the filter were detached by trypsin-EDTA and counted. All assays were performed in triplicate and the data expressed as percent inhibition by the peptides. Because caveolin oligomers are relatively insoluble in nonionic detergents, the reported association between β1 integrins and caveolin could possibly be an artifact of cellular disruption by detergents. Therefore, the distribution of β1 integrins and caveolin in cells in situ was assessed by immunostaining. Primary cultures of human vascular SMC were examined because these cells express relatively high amounts of β1 integrins (red) and caveolin-1 (green). Confocal microscopic analysis indicated these two proteins had marked spotty or array-like colocalizations over the cell body of SMC grown on glass coverslips . Substitution of isotype Ig controls for primary β1 and caveolin antibodies resulted in much weaker or no staining verifying the specificity of the immunostaining. Because β1 integrins and caveolin-1 only partially colocalize, we determined if β1 integrins and caveolin would cocluster. Clustering of SMC β1 integrins in suspension at 37°C resulted in the emergence of bright fluorescent signals of integrins (green) and caveolin (red) indicating clusters. Superimposition of the signals confirmed coclustering. No caveolin clustering was observed when the integrins were nonclustered or nonimmune Ig was substituted for the primary caveolin antibodies . These images indicate that fractions of β1 integrins and caveolin colocalize and are physically linked in cells before cellular disruption with detergents and are consistent with the hypothesis that caveolin has a role in integrin function. To explore more directly the importance of caveolin to β1 integrin function, parental 293 cells were transfected with antisense full-length mRNA for caveolin-1 under the influence of an inducible murine metallothionein promoter. Several clones with inducible suppression of caveolin protein expression were identified and examined. After 12–18 h of induction in the presence of 3 μM cadmium, two clones with normal caveolin levels, 6 and 9 , displayed strong adhesion to fibronectin . By contrast, two representative antisense clones, 31 and 32, which had 15– 50% the level of caveolin in parental 293 cells , displayed markedly impaired adhesion to fibronectin . Although 293 cells have only weak vitronectin adhesion, mediated by αvβ5 and αvβ1, this adhesion was also abrogated in caveolin-deficient cells (not shown). As is also indicated in Fig. 2 A, there was substantially less caveolin in these clones even without induction, indicating leakage of antisense expression, which has been reported for this expression vector in previous studies . Adhesion assays mirrored the extent of caveolin depletion: although they were capable of spreading on fibronectin, clones 31 and 32 were less adhesive even in the absence of induction . Nonetheless, clones 31 and 32 grew nearly as well as clones 6 and 9 over many months of passage. Flow cytometric analyses revealed no differences in surface level of β1 or αv or α5β1 heterodimers among these clones , indicating that depletion of caveolin expression affected the function rather than expression of β1 or its dimerization with α5 or other integrins. Caveolin antisense clones also exhibited time-dependent changes in cell shape with decreasing caveolin expression. Clones 6 and 31 were allowed to spread onto fibronectin for 1 h at 37°C, and then antisense caveolin mRNA induced. Within 8 h, clone 31 cells, but not clone 6 cells, became less spread. Within 18 h, clone 31 cells became round and numerous cells actually detached from the matrix. The shape changes paralleled loss of focal adhesion sites as indicated by loss of focal paxillin accumulation on ventral surfaces . In spite of the marked changes in adhesiveness and shape, most cells were found to respread and adhere better within hours of removing cadmium. We next considered mechanisms by which caveolin could influence integrin function. Because integrin expression per se was not changed in caveolin-depleted cells , the capacity of the various clones to bind 125 I-fibronectin in the absence and presence of a stimulating antibody (TS2/16) known to promote fibronectin binding by the major fibronectin receptor, α5β1, was determined . After induction, clones 6 and 31 were found to bind fibronectin equally at 4°C and exposure of the cells to TS2/16 enhanced fibronectin binding to a similar degree , indicating that the affinity states of α5β1 in control and caveolin-depleted clones are similar. Further, when surface β1 integrins were cross-linked by antibodies, the β1 integrins of normal (clone 6) and caveolin-depleted cells (clones 31 and 32) clustered equally well as judged by confocal microscopic analysis (not shown). In spite of the capacity of β1 integrins on caveolin-depleted clones to engage ligand, the changes in cell shape and adhesiveness provoked by caveolin depletion could not be reversed by the stimulatory β1 integrin antibody (TS2/16). These data imply that a postreceptor binding event(s) important to adhesion, rather than binding per se, is most likely impaired in caveolin-depleted cells. Src family tyrosine kinases are required for fibronectin receptor α5β1 and vitronectin receptor αvβ5-mediated cell adhesion . Moreover, tyrosine phosphorylation of FAK and other signaling molecules important to cytoskeletal reorganization and cell spreading after ligand engagement of β1 integrins may depend on interactions between FAK and Src family kinases . Because caveolin has been shown previously to interact with c-Src , it is possible that integrin-mediated tyrosine phosphorylation depends on caveolin. To test this hypothesis, we initially treated 293 cells with the tyrosine kinase inhibitor genestein (100 μM) and noted that 293 cells failed to attach and spread on fibronectin (data not shown). When β1 integrins were clustered with antibodies or 293 cells were allowed to adhere to fibronectin, increased FAK phosphorylation was apparent . In contrast, 293 cells with suppressed caveolin expression failed to exhibit FAK phosphorylation whether in response to antibody-induced clustering or ligand binding. This defect was not a general defect in tyrosine phosphorylation as autophosphorylation of ErbB-2, an EGF receptor–like tyrosine kinase, after binding of EGF, was not different between control and caveolin-deficient clones . The β1 integrins were next examined for interaction with nonreceptor tyrosine kinases by coprecipitation. Caveolin depletion of 293 cells (clones 31 and 32) resulted in loss of caveolin and Src family kinases in the β1 immunoprecipitates . The antibody used in this experiment was a pan-Src antibody recognizing multiple members of the src gene family. In additional experiments using specific antibodies we found c-Src, Fyn, and Yes in the β1 immunoprecipitates, all of which were almost completely lost in the β1 immunoprecipitates of caveolin-depleted cells (not shown). A major substrate for Src kinase, cortactin, implicated in adhesion and migration , was also markedly depleted in these precipitates. In situ kinase assays of β1 immunoprecipitates confirmed markedly reduced activity in cells (clones 31 and 32) depleted of caveolin . Although little or no Src family kinase or cortactin associates with β1 integrins in caveolin-depleted cells , this was not because tyrosine kinases could not be activated in these cells. Rather, kinase assays of immunoprecipitated c-Src confirmed a two- to fourfold increase in total c-Src activity in the caveolin-deficient clones (not shown). Previously, caveolin levels have been linked inversely to cellular Src kinase activity . Enhanced total kinase activity correlated with a striking hyperphosphorylation of cortactin in the caveolin-depleted clones (31 and 32) compared with the control clone 6 . Immunoblots of the cortactin immunoprecipitates revealed increased Src family kinases associated with tyrosine-phosphorylated cortactin, possibly through Src SH2 domain . These data indicate that caveolin-deficient 293 cells have dysregulated Src family kinase activity and little or no tyrosine kinase activity associated with their β1 integrins. uPAR forms complexes with β1 and β2 integrins and such complexes are important to uPAR-dependent adhesion to matrix vitronectin via a vitronectin binding site on uPAR . Caveolin is also found in uPAR/β1 integrin complexes. To explore the functional importance of caveolin to these complexes, we created stable cotransfectants of 293 cells expressing both uPAR and the antisense full-length mRNA for caveolin-1. Several clones with inducible suppression of caveolin expression were again identified. Control clones expressing uPAR but without reduction in caveolin levels adhered avidly to vitronectin . Clones with <50% the normal level of caveolin protein (clones m2–m5) adhered weakly or not at all to vitronectin even though uPAR and β1 integrin surface expression was not changed as judged by FACS ® analysis (data not shown). Immunoprecipitation of β1 integrins coprecipitated uPAR in proportion to caveolin levels and the ability of the cells to adhere to vitronectin , indicating that caveolin is required for stable uPAR/ integrin complexes and vitronectin adhesion. As with nontransfected 293 cells , depletion of caveolin in uPAR/293 cells resulted in almost complete loss of Src family kinases from the β1 integrin immunoprecipitates . As expected, the caveolin-depleted uPAR/293 cells also failed to adhere to fibronectin (not shown). In prior work, we observed that incorporation of α5/β1 integrins into complexes with uPAR, while stabilizing caveolin/integrin interactions, inhibited the ability of these integrins to bind fibronectin. Indeed, uPAR-transfected 293 cells had impaired fibronectin adhesion . In light of the critical role for caveolin in 293 cell adhesion to fibronectin and the fact that 293 cells have relatively low levels of caveolin, we considered the possibility that uPAR expression also inhibited β1 integrin function indirectly by sequestering caveolin into uPAR/ integrin complexes. Therefore, we transfected uPAR/293 cells with wild-type caveolin-1 and assessed the capacity of these transfectants to adhere to vitronectin and fibronectin. Concurrent caveolin overexpression in uPAR/293 cells had no effect on uPAR-dependent adhesion to vitronectin but completely restored the capacity of these cells to adhere to fibronectin . These data suggest competition for limited caveolin between unbound β1 integrin and uPAR/β1 integrin complexes may largely explain the impaired fibronectin adhesion of uPAR-transfected 293 cells. To explore further the functional connection between caveolin and uPAR in assembly of integrin-associated signaling molecules, lysates of uPAR/293 cells were immunoprecipitated for β1 integrins in the presence of either a peptide (peptide 25) which disrupts uPAR/integrin associations or a peptide derived from the “scaffolding domain” of caveolin-1 (cav-1). In experiments using purified proteins, cav-1 disrupts self-oligomerization of caveolin and blocks direct interactions between caveolin and other proteins, including c-Src . Both peptide 25 and cav-1 blocked coprecipitation of caveolin with β1 integrins in lysates prepared from either uPAR/293 cells or primary human vascular SMC . Both peptides, but not inactive control peptides, also blocked coprecipitation of Src family kinases and cortactin with β1 integrins , verifying that the stable association of these cytoplasmic proteins with integrins is dependent upon the membrane proteins caveolin and uPAR in these cells. Neither control peptide (peptide 36 and cav-x) had any effect on caveolin, cortactin, or Src coprecipitation with β1 integrins when compared with a no peptide control . Further, peptide 25 had no effect on coprecipitation of caveolin (or Src kinases) with β1 integrins in nontransfected 293 cells (not shown), confirming the specificity of this peptide for uPAR. Vascular SMC are known to express uPAR and β1 integrins and we confirmed uPAR expression by immunostaining. Clustering of β1 integrins resulted in uPAR coclustering, though no uPAR was detectable in these β1 immunoprecipitates by immunoblotting (data not shown). Although complex formation of β1 integrins with uPAR could be expected to inhibit fibronectin adhesion , SMC attach and spread well on fibronectin, mostly via α5β1. As discussed above, this may be explained by the relatively high levels of caveolin in SMC as compared with 293 cells. Nonetheless, peptide 25 but not a control peptide, was found to disrupt caveolin and Src kinase associations with β1 integrins almost completely in lysates of SMC . Therefore, we used this peptide to explore the role of caveolin in the integrin function of intact SMC. Incubation of SMC adherent to fibronectin with peptide 25 before lysis resulted in loss of Src kinases (as well as caveolin) in the β1 immunoprecipitates . Consistent with the loss of signaling molecules, integrin-dependent MAPK activation was also blocked by peptide 25 . It should be noted that the pathway of integrin and uPAR– dependent MAPK activation in these cells is not yet defined. Although MAPK is reproducibly phosphorylated within 30 min of fibronectin attachment, we can detect no Shc or FAK phosphorylation (not shown). The inhibitory effects of the peptide on association of Src family kinases with integrins was correlated with loss of integrin function. Although neither peptide blocked adhesion, peptide 25 delayed SMC spreading on fibronectin and blocked migration of human SMC across microbore filters to fibronectin-coated surfaces , consistent with its effect on integrin signaling. Expression of the capacity of integrins to mediate cellular adhesion has several distinct facets: (a) the conformational state of integrins, affecting the affinity of ligand binding; (b) ligand-induced integrin clustering, enhancing the strength of attachment and promoting interactions between kinases and their substrates important to signal transduction; and (c) signaling itself, activating a cascade of events leading to organization of the cytoskeleton and cell spreading . Numerous in vitro studies and recent evaluations of mice with targeted deletions in nonreceptor tyrosine kinases implicate FAK and Src family kinases as key mediators of integrin signaling . Thus, the molecular events leading to assembly and activation of these kinases surrounding integrins are important determinants of integrin function. This conclusion has focused much attention on nonintegrin membrane proteins which might regulate this process and our data identify one such protein as caveolin. While caveolin does not affect integrin expression or the intrinsic capacity of β1 integrins to bind fibronectin , caveolin is required for the normal assembly of adhesion plaques which develop in response to ligand engagement and integrin clustering. In the absence of sufficient β1 integrin–associated caveolin, there is loss of integrin-associated Src kinase activity, little or no FAK activation after ligand binding, and impaired association of integrins with structural proteins such as tyrosine-phosphorylated cortactin important to β1 integrin– dependent adhesion . As a result, the characteristic accumulation of enzymes and structural proteins which comprise integrin-dependent adhesion sites fails to develop . Our results confirm and extend the recent findings of Wary and colleagues that caveolin expression is required for the association of Fyn kinase with β1 integrins and ligand-dependent Shc phosphorylation. These investigators began with cells (Fisher rat thyroid cells) expressing little or no caveolin or α5 integrin and transfected both proteins. They found that physical association of the Src family kinase Fyn with α5/β1 required concurrent coexpression of caveolin-1. Fyn but not Src was required for α5/β1-dependent Shc phosphorylation. Beginning with cells having functional β1 integrins and expressing caveolin-1, we suppressed caveolin expression and found β1 integrin association with several Src family kinases to be completely dependent on caveolin. In both cases, deficient β1 integrin–associated Src family kinase activity led to correspondingly marked changes in integrin function. Together, these findings indicate caveolin is a general regulator of β1 integrin function. How does caveolin promote integrin signaling? Although our understanding is incomplete, we favor a model in which caveolin functions primarily to sequester Src family kinases in an inactive configuration at sites proximate to integrins, promoting their presentation to integrins and activation during ligand-induced integrin clustering. Caveolin has been shown previously to interact predominantly with the inactive form of Src and to reduce Src activity when overexpressed in 293 cells, perhaps by sequestering Src kinases from activating phosphatases and substrates . Consistent with this model, caveolin-deficient 293 cells were found to have two- to fourfold higher levels of basal c-Src kinase activity (not shown) and to exhibit striking hyperphosphorylation of a major Src substrate, cortactin . If one function of caveolin is to suppress basal Src kinase activity, the model in Fig. 7 also offers a possible molecular explanation of how caveolin contributes to Src family kinase activation as a consequence of integrin clustering. Caveolin interactions with Src family kinases are thought to occur through the same membrane proximal sites on caveolin as those involved in formation of caveolin homo-oligomers . Because coclustering of caveolin with integrins could be expected to favor caveolin homo-oligomer formation, integrin clustering is likely to modify the physical interaction of caveolin with Src kinases. This may lead to outright release of these kinases from caveolin and/or an altered, more accessible position of these kinases in the cluster favoring their activation through contact with substrate or through a phosphatase. In either case, these studies directly implicate caveolin function in the membrane proximate events of ligand-induced, β1 integrin–dependent tyrosine kinase activation. As caveolin levels are reportedly low in a number of transformed cells , our data raise the possibility that low caveolin levels contribute to the defective fibronectin adhesion and matrix assembly seen in many cancer cells and thought to be important to their metastatic potential . A further implication of the studies reported here and the recent studies of Wary and colleagues is that Fyn and Src may have overlapping but distinct roles in β1 integrin signaling: Fyn being primarily required for Shc phosphorylation and activation of an MAPK-dependent growth pathway and Src being primarily required for FAK and cytoskeletal phosphorylation and assembly of focal adhesion sites. This concept is supported by results of prior studies examining mice deficient in the negative regulator of Src family kinase activity, Csk . Fibroblasts from Csk− embryos exhibit hyperphosphorylation of adhesion sites and striking hyperphosphorylation of cortactin, reminiscent of that seen in caveolin-deficient cells . The hyperphosphorylation of adhesion sites and cortactin was largely corrected by crossing the Csk− mice with Src− mice but not by crossing with Fyn− mice, again implicating Src as primarily being involved in regulation of integrin-dependent adhesion. Clearly this is an oversimplification since we also observe Yes as well as Fyn and Src in β1 integrin/caveolin complexes and even kinase-inactive Src can promote focal contact assembly in Src− cells . Still, these recent studies support the possibility that different members of the src gene family have favored roles in integrin signaling even in cells expressing multiple family members. The structural basis for the association between caveolin and β1 integrins is uncertain. First, most if not all cellular caveolin is reportedly found in purified caveolae . Although integrins have not been observed in purified caveolae, when we subject 293 cells to mechanical lysis, sonicate, and centrifuge the postnuclear supernatant through a sucrose gradient, caveolin in 293 cells appears at the 5/35% sucrose interface, typical for that reported for caveolae preparations in 293 and other cells . Almost all of the β1 integrins appear in the fractions containing caveolin (Yang, X., unpublished observation). These findings are consistent with the scenario that β1 integrin/ caveolin/Src family kinase complexes exist as signaling units in caveolae and separate from caveolae in response to ligand-induced clustering and cytoskeletal reorganization. However, the exact composition of caveolae remains controversial and appears strongly dependent on the method of purification . An alternative possibility is that some fraction of caveolin traffics outside caveolae, as has been argued by Wary and colleagues . This will be an important issue for future experiments. In either case, the fraction of β1 integrins which colocalize and cocluster with caveolin appears to be critical to β1 integrin signaling. Whether β1 integrins directly bind caveolin is also uncertain. Wary and colleagues reported that the α5 transmembrane domain was required for coprecipitation of α5/β1 and caveolin. However, no direct binding has been demonstrated. Most if not all other proteins that are reported to bind caveolin bind sequences of caveolin that are membrane proximal but in the cytoplasmic compartment . Indeed, in SMC we observed a peptide comprised of a membrane proximal region of caveolin (amino acids 82–101) blocking coprecipitation of caveolin with β1 integrins . Thus, it is uncertain whether β1 integrins and caveolin directly bind or associate indirectly through a common affinity for certain membrane lipids or a third protein such as uPAR which may directly interact both with integrins and cholesterol-rich lipid domains enriched in caveolin. The physical basis for the association of β1 integrins and caveolin also requires further investigation. The marked inhibition of β1 integrin function by uPAR-binding peptides in human SMC is remarkable. Although we could detect uPAR in SMC by immunostaining and could cocluster uPAR along with β1 integrins, no uPAR was detected in the β1 immunoprecipitations of SMC. This suggests the association of uPAR with β1 integrins and caveolin in these cells is relatively weak. There also appears to be a stoichiometric excess of β1 integrins compared with uPAR in SMC. Thus, it is surprising that the coprecipitation of most caveolin- and integrin-associated Src kinases with β1 integrins would be blocked by peptide 25, raising the possibility of an in vitro artifact. This is an unlikely explanation for the findings because the uPAR-binding peptide (peptide 25), but not two control peptides (peptide 36 and a scrambled version of peptide 25), also had clear biochemical and functional effects on intact SMC. Prior treatment of SMC with the active peptide blocked Src kinase association with β1 integrins and inhibited both signaling and migration of the cells on fibronectin . These results indicate that, although not intrinsically required for the function of integrin/caveolin , the presence of uPAR organizes caveolin and its associated signaling molecules in an interdependent manner such that integrins, caveolin, and uPAR form a unit promoting integrin function. Perhaps this is possible because only a fraction of β1 integrins associates with caveolin and uPAR associates mainly with activated integrins . As indicated in Fig. 7 , integrin activation initiated by ligand-induced integrin clustering may promote formation of these signaling complexes. Similar mechanisms may account for observations that uPAR is required for normal function of the β2 integrins, CD11/ CD18 , and for migration on vitronectin mediated by αvβ5 , suggesting a promoting effect of uPAR on integrin function may be a widespread phenomenon. | Study | biomedical | en | 0.999998 |
10087271 | Primary myoblasts were isolated from pectoralis muscle of nine day Japanese quail embryos as previously described . In brief, the breast muscle was dissected from the embryo and myoblasts were dissociated from muscle tissue with 0.1% dispase ( Sigma Chemical Co. ) in PBS. The cell suspension was filtered through a Sweeney filter; cells were seeded onto gelatin-coated tissue culture plates (0.1% gelatin in PBS). Myoblast cultures were maintained in complete myoblast medium (DMEM [ Sigma Chemical Co. ] containing 15% horse serum, 5% chick embryo extract, 1% pen/strep, and 1.25 mg/ml fungizone [ GIBCO BRL ]). Myoblasts were subcultured in trypsin-EDTA (0.06% trypsin, 0.02% EDTA) and used between passages 1 and 10. The muscle α-actinin–specific mAb, 9A2B8, was kindly provided by D. Fishman (Cornell University, New York, NY) as a hybridoma supernatant. mAb VIF4, which recognizes the human α5 integrin extracellular domain was a gift of R. Isberg (Tufts University, Boston, MA). The chicken α6-specific polyclonal antibody, α6ex , was provided by L. Reichardt (University of California, San Francisco, CA). mAb 2B7 directed against the extracellular domain of the human α6 integrin , was a gift of A. Mercurio (Harvard Medical School, Boston, MA). The mAb 165 is directed against paxillin . mAb 2A7 directed against FAK and the polyclonal Ab BC3 directed against FAK were gifts of T. Parsons (University of Virginia, Charlottesville, VA). The anti-FAK polyclonal antibody C-20, was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-active MAPK polyclonal antibody, which specifically recognizes dually phosphorylated, activated MAPK, was purchased from Promega . The anti-MAPK mAb that recognizes erk-1, as well as the anti-phosphotyrosine antibodies, RC20H and PY20, were purchased from Transduction Laboratories. The anti-hemagglutinin (HA) mAb, 12CA5, was purchased from Boehringer Mannheim . The anti-human CD2 mAb, TS2/18.1.1 was purchased from the Developmental Studies Hybridoma Bank. TS2/16, a mAb against human β1 integrin was from M. Hemler (Dana-Farber Cancer Institute, Boston, MA). Antibody against the human IL2 receptor was purchased from Boehringer Mannheim . Poly- l -lysine was purchased from Sigma Chemical Co. Fibronectin was purified from human plasma by affinity chromatography as previously described . Laminin was isolated from murine Englebreth-Holm-Swarm sarcoma as previously described . The human α5 cDNA in pRSVneo and the chicken α6 cDNA in pRSVneo were described previously . The chicken α61044t truncation was constructed by first subcloning a 1.6-kb HindIII-SalI fragment of the chicken α6A DNA into M13 and then introducing an in-frame BclI site at amino acid position 1044 . Mutants were confirmed by restriction digestion of M13 clones with BclI and by single stranded DNA sequencing using the dideoxy-chain termination method according to the Sequenase™ protocol (United States Biochemical Corp.). An 800-bp BstXI-SalI fragment containing the mutation was subcloned into pRSVneoα6 partially digested with SalI and completely with BstXI. The human α6A and α6B cDNAs, in the expression plasmid pRc/ CMV , were a generous gift of A. Mercurio (Harvard Medical School, Boston, MA). The pRSVneo-CH8β1 plasmid was constructed by subcloning a 1-kb HindIII fragment, containing the CH8 epitope tag, from the CH8β1 pBJ-1 construct received from Y. Takada (Scripps Research Institute, La Jolla, CA) into pRSVneoβ1 expression vector . pRSVIL2R-α5 and pRSVIL2R-β1A were constructed by cloning an Nhe1-Xba1 fragment from pCMVIL2R-α5cyto or pCMVIL2R-β1A plasmids received from Susan LaFlamme into the Xba1 site of the pRSVneo vector. Clones were screened for orientation by restriction digests. HA-tagged rat MEK1 and HA-tagged rat constitutively active (CA) MEK S218/220D in pCMVneo vector i.e., sodium-deoxycholate, sodium-pyrophosphate, sodium-orthorandate were received from M. Weber (University of Virginia, Charlottesville, VA). The chicken paxillin cDNA, the Y118F, and the S188/ 190A mutants in the pcDNA3.0neo vector were received from C. Turner. CD2FAK, CD2FAK(Y397F), and CD2FAK(K454R) in CDM8 vector were received from A. Aruffo. Cells were transiently or stably transfected using a liposome-DNA solution as previously described previously . In brief, replicating myoblasts (passages 1–3) were plated on 60-mm tissue culture plates coated with 0.1% gelatin in complete myoblast medium for 16–20 h. Cells were incubated for 8–16 h in a solution of 8 μg of plasmid DNA and 50 μg of Lipofectamine ( GIBCO BRL ) in complete myoblast medium. Transfected myoblasts were either washed with DMEM, refed with myoblast medium, and analyzed for transient expression or were trypsinized and plated into selection medium on gelatin-coated tissue culture plates (myoblast medium containing 0.4 mg/ml G418; GIBCO BRL ) for 7–12 d and then into myoblast medium containing 0.2 mg/ml G418 (maintenance medium). For transfections with CD2FAK constructs, in order to generate stable populations, myoblasts were cotransfected with a pRSVneo or a pEGFP-C1neo plasmid ( Clontech , Palo Alto, CA) at 1:7 ratio (neo resistance gene:CD2FAKcDNA) and selected in G418 as previously described. The chicken α6, human α6A or α6B, and α61044t transfections were selected and maintained on laminin-coated (20 μg/ml) tissue culture plates. For coexpression of hα6A integrin and CD2-FAK cells were cotransfected with pRc/CMVhα6A and CDM8CD2FAK vectors at a ratio of 1:7, respectively. Transiently transfected cells were sorted by flow cytometry (see below) for hα6A expression and the positive cells were grown in G418 containing medium. CDM8CD2FAK vector does not carry a neo resistance gene, therefore, only cells carrying both neo resistance (hα6A positive) and able to replicate (CD2FAK positive) will survive. Stable populations were analyzed both for hα6 and CD2 expression as described below. Both transiently and stably transfected (the α5 phenotype was seen in transient as well as stable transfectants) myoblasts were analyzed for surface expression by flow cytometry as previously described . Chicken α6A and α61044t transfected cells were stained with a chick α6-specific polyclonal antibody, α6ex, at 20 μg/ml in blocking buffer (Hepes-Hanks PBS-CMF with 2% BSA) and FITC-labeled goat anti–rabbit IgG (Cappel). Human α6A or B transfected myoblasts were analyzed with the human α6-specific mAb, 2B7, at 10 μg/ml in blocking buffer. Human α5 transfected cells were stained with VIF4 mAb. Cells transfected with CD2FAK or its mutants were stained with anti-CD2 mAb TS2/ 18.1.1. Chicken β1 transfected cells were stained with TS2/16 mAb against the human β1 epitope. IL2R-α5 or IL2R-β1A transfected cells were analyzed with an anti-IL2R antibody. The FACS profiles of the IL2R-α5 and IL2R-cyto-transfected cells were not stable, and we were unable to obtain populations greater than 40% positive, which were used for analysis. Flow cytometry was performed on a EPICS cell sorter (Coulter Electronics, Inc.) equipped with Cicero software for data analysis. As shown in Fig. 1 , expression levels were assayed by fluorescence activated cell sorting. These profiles reflect enriched surface expression levels of ectopic integrin subunits in transfected myoblasts used in experiments. For Western blotting and immunoprecipitation experiments, untransfected and transfected myoblasts were plated on FN (UT and hα5 transfected cells), on LM or on gelatin (UT, PAX, and CD2FAK transfected cells) for 24 h in complete myoblast medium. Since myoblasts will differentiate in the absence of serum and also secrete their own matrix, we were unable to test the effects of specific matrix ligands on the myoblast response. Therefore, all assays were conducted in the presence of serum and results are presented for steady state conditions. Cells were washed with ice-cold PBS containing 1 mM Na-orthovanadate and lysed in ice-cold modified RIPA extraction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 1.0% Triton X-100, 0.25% sodium-deoxycholate, 2 mM EDTA, and 2 mM EGTA) with protease inhibitors (20 mg/ml leupeptin, 0.7 mg/ml pepstatin, 1 mM phenanthroline, 2 mM phenyl-methyl-sulfonyl-chloride, and 0.05 units aprotinin) and phosphatase inhibitors (30 mM sodium-pyrophosphate, 40 mM NaF, 1 mM sodium-orthovanadate). Protein content of the clarified lysates was determined using the Pierce bicinchoninic acid (BCA) method with bovine serum albumin as the standard. For phosphotyrosine Western blots, 10–15 μg of lysates were separated on 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose membranes . Membranes were blocked in 1% heat denatured BSA in TST buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20) overnight at 4°C. Phosphotyrosine containing proteins were detected by incubating the membranes with the anti-phosphotyrosine mAb, PY20, and a secondary horse radish peroxidase (HRP) conjugated anti–mouse antibody (Jackson ImmunoResearch Labs) or with RC20H, a directly conjugated HRP anti-phosphotyrosine Ab. Blots were visualized by chemiluminescence ( Pierce Chemical Co. ). Membranes were exposed to X-ray film ( Kodak , X-OMAT AR) and developed in an automatic film processor. When indicated, membranes were stripped in stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β-mercaptoethanol) for 30 min at 60°C and reprobed with a different antibody. For anti-MAPK Western blots, cells were trypsinized, washed once with soybean trypsin inhibitor (0.5 mg/ml), washed twice in Puck's Saline G ( GIBCO BRL ) and resuspended in serum-free medium containing 2% BSA. Cells were held in suspension for 1 h prior to plating on FN or LM in complete myoblast medium for 24 h. Cell extracts were prepared in RIPA buffer as described. 5 μg of cell lysates were separated on 12% SDS-PAGE gels under reducing conditions and the proteins transferred to nitrocellulose membranes. The membranes were blocked in 3% nonfat dry milk in TST overnight at 4°C. Active MAPK was detected by an anti-active MAPK pAb ( Promega ). Membranes were stripped and reprobed for total MAPK with an anti-erk1 mAb (Transduction Labs) or SC-94 anti-erk1 pAb ( Santa Cruz Biotech nologies). For paxillin, FAK, CD2-FAK, and HA immunoblot analysis, 5–20 μg cell lysates were resolved on 7.5% SDS-PAGE gels under reducing conditions and proteins transferred to nitrocellulose membranes. Membranes were blocked in TST buffer containing 3% nonfat milk and the proteins were detected with 165 mAb (anti-paxillin), BC3 pAb (anti-FAK), TS2/ 18.1.1 mAb (anti-CD2), or 12CA5 mAb (anti-HA). For FAK immunoprecipitations, 100 μg of RIPA lysate was mixed with 1 μl of anti-FAK mAb, 2A7, 50 μl of packed agarose anti–mouse beads (blocked in 5% BSA; Sigma ) in a final volume of 500 μl. The bead-antibody-antigen complex was incubated at 4°C for 2 h with continuous agitation. For paxillin immunoprecipitations, 100 μg of cell lysate and 1 μl of anti-paxillin mAb, 165 were incubated at 4°C with continuous agitation for 1 h. In a separate tube, 50 μl of packed protein A–agarose beads and 30 μg/ml rabbit anti–mouse IgG were incubated in lysis buffer for 1 h. The antigen-antibody mixture was then added to rabbit anti–mouse–protein A beads and incubated at 4°C an additional 2 h. The beads were pelleted gently and washed twice with lysis buffer. Bound protein was released from the beads by boiling in 100 μl Laemmli sample buffer containing 5% β-mercaptoethanol for 5 min. Equal aliquots of the precipitated protein for each antibody were loaded onto 7% SDS-PAGE gels. The FAK IP was blotted for FAK with C-20 and for phosphoFAK with RC20H. The paxillin IP was blotted for paxillin with the 165 mAb or for phosphopaxillin with RC20H. All immunoprecipitations and Western blots were detected by chemiluminescence. Cells were grown on FN- or LM-coated coverslips. Immunostaining was done at room temperature. Cells were rinsed in PBS and fixed with 3% formaldehyde in PBS for 15 min then permeabilized with 0.4% Triton X-100 in PBS for 10 min, washed and blocked in 5% goat serum in PBS (BB) for 30 min. Cells were incubated with primary Ab in BB for 30 min, washed and incubated with FITC- or rhodamine-conjugated secondary Ab (Cappel) and DAPI ( Sigma Chemical Co. ) for additional 30 min. Coverslips were washed extensively and mounted in medium containing elvanol and p -phenylenediamine. Fluorescence was observed on a Zeiss Axioplan microscope. MAP kinase activity was manipulated in hα6A transfected myoblasts by coexpression of constitutively activate (CA) MEK1. Myoblasts were cotransfected with pRc/CMVhα6A and the HA-tagged pCMVneoMEK S218/220D vectors at a ratio of 1:7, respectively. Cells were selected in G418 and stable populations were sorted by flow cytometry for human α6A expression as described. Cell lysates were analyzed for HA expression by Western blotting as described. Stably cotransfected cells were plated on LM-coated plates and observed for 96 h. To alter MAP kinase activity in hα5 transfected myoblasts, hα5-expressing cells were grown in the presence of the specific MEK inhibitor PD98059 ( New England Biolabs ) . Transfected myoblasts were plated on FN-coated coverslips and on FN-coated TC plates. After 8 h in complete myoblast medium, the first dose of the inhibitor was added to the cells at 1, 10, 25, 50, or 100 μM final concentration. Cells were grown for an additional 24 h and a second dose of the inhibitor was added. After 24 and 48 h in presence of the inhibitor, coverslips were fixed and immunostained for DAPI and muscle α-actinin. At the same time, cells were extracted in RIPA buffer as described and lysates were analyzed by Western blotting for active MAPK and total erk1 expression as described above. Differentiation was scored using the fusion index, which is the percentage of total nuclei in myotubes as described in Sastry et al. . We recently reported a specificity for integrin α subunits and their cytoplasmic domains in controlling the proliferative to differentiative transition in primary quail myoblasts . Ectopic expression of the human α5 integrin subunit (hα5) enhanced the fraction of myoblasts remaining in the proliferative phase and inhibited the initiation of terminal differentiation. In contrast, ectopic expression of the human α6A subunit of integrin (hα6A) inhibited myoblast proliferation and promoted differentiation. These effects resulted from a three- to fivefold increased surface expression of the α5β1 or the α6Aβ1 integrin (and a two- to threefold increase in total β1 integrin, see below) with little change in the relative expression of other integrin α subunits. These findings suggested that the α5 cytoplasmic domain promotes proliferative signals whereas the α6A cytoplasmic domain inhibits proliferation and enhances the fraction of cells initiating terminal differentiation. To assess the contribution of these two cytoplasmic domains, we first examined the effect of ectopic α5 and α6A truncation mutants on myoblast proliferation and differentiation. . As we reported previously, ectopic expression of the hα5 truncation, α5GFFKR, which retains only the conserved GFFKR sequence, promoted proliferation and inhibited differentiation similar to the wild-type hα5 subunit . These findings suggest that the majority of the α5 cytoplasmic domain is not required for proliferative signals. On the other hand, ectopic expression of an α6A truncation, α61044t, which deletes the COOH-terminal 11 amino acid residues, restores proliferative signaling and produces a phenotype similar to that of the ectopic α5 subunit. Myoblasts expressing α61044t remain in the proliferative phase and do not differentiate even in high density cultures . Like hα5 transfected myoblasts , myoblasts expressing α61044t do not express muscle α-actinin , a myogenic differentiation marker, and exhibit a fusion index of 5% after 72 h of culture in a rich medium. This contrasts UT controls and hα6A transfected cells where a significant fraction of cells express muscle α-actinin and fuse into multinucleated myotubes . Preliminary mapping of the COOH-terminal 11 amino acids points to S1071 (in hα6A) as a key residue, since its mutation to alanine produces a phenotype with enhanced proliferation (data not shown). Furthermore, the proliferation inhibiting effect of hα6 integrin is specific for the α6A cytoplasmic domain isoform. Ectopic expression of the hα6B subunit in myoblasts promotes proliferation and inhibits differentiation (data not shown, Table I ). Consistent with these results, the α6A isoform is the predominant α6 integrin expressed in striated muscle and in embryonic cells of determined lineage whereas α6B is highly expressed in proliferating, totipotent or undifferentiated ES cells. Taken together, these observations suggest that the α5 and α6A cytoplasmic domains function differently: the α5 cytoplasmic domains appears permissive whereas a discrete region of the α6A cytoplasmic domain is inhibitory with respect to proliferation. To determine whether the α5 or α6A cytoplasmic domains act directly or indirectly, we assayed the effects of single-subunit cytoplasmic domain chimeras , IL2R-α5 or IL2R-α6A, on the ability of myoblasts to proliferate or differentiate. Ectopic expression of either IL2R-α5 or IL2R-α6A had little detectable effect on myoblast proliferation or differentiation . These cells behaved much like control, untransfected (UT) myoblasts. Thus the α subunit cytoplasmic domains do not directly initiate signals for myoblast proliferation or differentiation. How then do these two integrin α subunits regulate proliferation and differentiation? Our observation that different integrins, e.g., α5, α6B, and α61044t, all produce a similar phenotype suggests an hypothesis in which these α subunits influence the proliferative signaling through the β1 subunit. In this view the α5 cytoplasmic domain would permit signaling through the β1 subunit whereas the α6A cytoplasmic domain would inhibit it. Previous studies have shown that the α subunit cytoplasmic domain can regulate β1 integrin localization to focal adhesions and integrin activation ; both localization and activation, however, are mediated by the β subunit cytoplasmic domain. Furthermore, the β1 cytoplasmic domain alone, when expressed as a single subunit chimera, IL2R-β1, can activate intracellular signals . To test this hypothesis, we first determined whether overexpression of the chicken β1A subunit of integrin would increase the fraction of proliferative myoblasts. We chose the β1A isoform since it is predominant in replicating myoblasts . As reported in Sastry et al. ectopic expression of integrin α subunits also produces a two- to threefold increase in total β1 expression with little change in relative expression of the other endogenous α subunit levels. The increase in total β1 expression maintains myoblasts in the proliferative phase and inhibits terminal differentiation. Myoblasts with enhanced β1A expression grow to confluency but exhibit a fusion index of only ∼10% compared with 60–70% for untransfected cells . Thus, increased β1A expression produces a phenotype resembling that of increased hα5 subunit expression. This finding agrees with a similar result reported previously for the β3 integrin . Next, we addressed whether the β1A cytoplasmic domain could independently affect myoblast proliferation or differentiation through ectopic expression of the single subunit chimera, IL2R-β1A. Previous studies show that this chimera localizes in focal adhesions and mediates enhanced integrin signaling . Myoblasts expressing IL2R-β1A remain replicative and proliferate until confluent with little detectable fusion into myotubes . Like myoblasts expressing ectopic α5 subunit, they also only exhibit significant cell cycle withdrawal and differentiation if cultured under serum-free conditions . These results demonstrate that the β1A cytoplasmic domain is sufficient to transmit proliferative signals and inhibit differentiation and thus modulate the growth factor response. Further, ectopic expression of IL2R-β1A can rescue the hα6A phenotype. Myoblasts that coexpress IL2R-β1A and hα6A integrins proliferate and differentiate like untransfected cells (data not shown; Table I ). Taken together, these findings suggest that proliferative signaling through integrins occurs via the β1 subunit and that different α subunit cytoplasmic domains can modulate these signals. The effects of ectopic integrin subunits on myoblast proliferation and differentiation are summarized in Table I . We next sought to determine the effect of ectopic α5 or α6A expression on β1A integrin signaling pathways. Since integrins stimulate increased tyrosine phosphorylation of several intracellular proteins , we assayed the phosphotyrosine profile of myoblasts expressing different ectopic α subunits. Immunoblotting with an anti-phosphotyrosine antibody shows that myoblasts ectopically expressing the hα5 integrin contain elevated tyrosine phosphorylation of proteins migrating in the molecular mass ranges of 120–130 and 65–70 kD whereas cells transfected with hα6A show a marked, general decrease in tyrosine phosphorylation with no additional bands when compared with UT controls (data not shown). These observations are consistent with the phenotypic effects of the ectopic integrins presented above; i.e., the α subunits do not initiate separate pathways. Thus, ectopic α5 expression permits enhanced signaling through the β1A subunit, whereas the α6A integrin suppresses these signals. Next, we pursued the identities of the phosphoproteins migrating at 120–130 and 65–70 kD. Focal adhesion kinase (pp125FAK) and paxillin (pp68) are downstream targets of integrin signaling pathways that migrate in these molecular mass ranges . Therefore, we assayed the level of FAK and paxillin tyrosine phosphorylation in UT, hα5, and hα6A transfected myoblasts. A Western blot of a FAK immunoprecipitation shows that comparable levels of FAK were precipitated . The level of tyrosine phosphorylated FAK decreased in hα6A versus hα5 transfected or UT myoblasts . However, the level of FAK tyrosine phosphorylation in hα5 and UT myoblasts does not differ . Immunodepletion of FAK from the lysate, followed by Western blotting the supernatant for phosphotyrosine reveals an additional 120-kD band in the hα5 transfected cells that could account for the observed increase in phosphotyrosine (data not shown). Thus, whereas FAK phosphorylation decreases in myoblasts expressing ectopic α6A integrin, it is unaffected by ectopic α5 expression. To determine if paxillin phosphorylation is differentially regulated in UT, hα5, and hα6A transfected myoblasts, paxillin was immunoprecipitated with an anti-paxillin mAb. In contrast to observations with FAK, we observed a major difference in the level of paxillin expression between UT and hα5 transfected myoblasts. As seen in Fig. 3 B, paxillin is significantly upregulated in hα5 transfected myoblasts when compared to UT (lane 1) or hα6A transfected (lane 3) myoblasts. The comparable intensity of a 55-kD band in the paxillin immunoprecipitation corresponds to reduced IgG and serves as a loading control. In addition to elevated levels of paxillin, a phosphotyrosine Western blot of the paxillin immunoprecipitation shows a concomitant increase in tyrosine phosphorylation of paxillin in hα5 transfected myoblasts compared to UT myoblasts (lane 4). Tyrosine phosphorylation of paxillin in hα6A transfected cells (lane 6) is somewhat decreased relative to untransfected cells. The enhanced paxillin expression observed in hα5 transfected myoblasts does not arise as a direct effect of the hα5 integrin. Myoblasts expressing IL2R-β1A also show increased paxillin expression whereas myoblasts expressing IL2R-α5 do not (data not shown). Taken together, these results indicate that enhanced paxillin expression accompanies increased α5 (or β1A) levels, whereas decreased FAK phosphorylation coincides with increased α6A levels. These data also suggest an uncoupling of FAK and paxillin signaling. The altered expression and activation of paxillin and FAK presented above prompted us to examine the effects of ectopic paxillin or FAK expression on myoblast proliferation and differentiation. As shown in Fig. 4 A (lane 2), the level of paxillin expression increases when compared with controls after transfection of a paxillin cDNA. Ectopic expression of wild-type paxillin inhibits differentiation and results in a proliferative phenotype . Myoblasts expressing ectopic paxillin proliferate until confluent but neither fuse into multinucleated myotubes nor express muscle α-actinin . This phenotype is similar to that of hα5 transfected myoblasts . Thus ectopic paxillin expression alone can recapitulate the effects of the hα5 or IL2R-β1A integrin subunits. Paxillin expression levels in control cells do not differ in replicating myoblasts versus differentiated cultures (data not shown). Two major sites of phosphorylation in paxillin in response to adhesion to fibronectin are Y118 and S188/190 . The tyrosine phosphorylation site, Y118, is also the site phosphorylated by FAK . Therefore, we next tested the effect of a Y118F mutation or the double mutation S188/190A on the myoblast phenotype. When expressed in myoblasts , neither of these mutants showed the enhanced proliferation seen when wild-type paxillin was expressed. Instead the paxillin mutants exhibited a phenotype characteristic of UT myoblasts . This result suggests that these tyrosine and serine phosphorylation sites in paxillin participate in proliferative signaling that regulates myoblast cell cycle withdrawal. Since FAK phosphorylation decreased in parallel with the inhibition of proliferation in myoblasts expressing hα6A integrin, we next tested the effect of an ectopic FAK mutant, Y397F, which lacks the autophosphorylation site and cannot bind src-family kinases , on proliferation and differentiation of myoblasts. Since ectopic expression of soluble FAK often produces short-lived or weak phenotypes , we used CD2-FAK, a membrane bound, chimeric FAK construct, which is constitutively active . Presumably, this results from increased adhesive signaling that arises from its constitutive membrane association and consequent localization in focal adhesions. Ectopic expression of CD2-FAKY397F, inhibits myoblast proliferation while promoting differentiation . These cells are reminiscent of myoblasts transfected with the hα6A integrin subunit except that their proliferation is not inhibited as completely. They also show extensive fusion into multinucleated myotubes . Interestingly, myoblasts transfected with wild-type CD2-FAK , remain proliferative and do not initiate terminal differentiation when compared to UT controls. Fewer than 5% of CD2-FAK–expressing myoblasts fuse into multinucleated myotubes or express muscle α-actinin . Using FACS analysis of propidium iodide labeled cells to measure G1/S progression, CD2-FAK transfected myoblasts show an increased ratio of G2 to G1 cells compared to untransfected cells (data not shown). Similarly, ectopic expression of CD2-FAK454, which is kinase defective, also inhibits differentiation and promotes proliferation . Ectopic expression of wild-type CD2-FAK in hα6A transfected myoblasts results in a proliferative phenotype. Myoblasts that coexpress CD2-FAK and hα6A integrin grow to confluency and do not differentiate . In sum, the hα6A phenotype can be recapitulated by expression of a FAK mutant expressed in control myoblasts or rescued by coexpression of an activated form of FAK. These data suggest that one potential mechanism by which hα6A integrin inhibits myoblast proliferation is through altering FAK phosphorylation. Thus, changes in the level of focal adhesion signaling, through FAK or paxillin, significantly affect the likelihood of myoblasts to proliferate or withdraw from the cell cycle and differentiate. Table II summarizes the effects of ectopic focal adhesion molecules. Several reports implicate MAP kinase in adhesion dependent regulation of proliferation . In addition, MAP kinase activation plays an important role in muscle differentiation . Therefore, we investigated if the ectopic integrins altered MAP kinase activation to control myoblast proliferation and differentiation. MAP kinase activation was assessed by immunoblotting cell lysates with an antibody that specifically recognizes phosphorylated, or active, forms of the 42- and 44-kD MAP kinases. Quail myoblasts express the 44-kD MAP kinase, erk-1, which was detected using an anti–erk-1 mAb (data not shown). Fig. 6 shows Western blots of active MAP kinase in myoblasts expressing ectopic integrins. In all cases, control or transfected cells were cultured for 24 h in complete serum-containing medium before extraction. The results shown reflect differences in steady state levels of MAP kinase activity. Compared with UT myoblasts , hα5 (lane 2), or IL2R-β1A (lane 3) transfected myoblasts contain elevated levels of active MAP kinase. In contrast, the level of active MAP kinase in hα6A transfected cells is significantly decreased compared with controls (top, lane 1). The different intensities for control levels of active MAP kinase (top versus bottom) is due to different exposure times of the Western blots to film. As presented earlier, the α6A cytoplasmic domain truncation, α61044t, did not produce the proliferation inhibiting effects of hα6A integrin, and instead promoted proliferation and inhibited cell cycle withdrawal. This also altered the level of MAP kinase activation, as assayed by Western blotting. Compared with myoblasts expressing hα6A, those expressing α61044t display enhanced levels of active MAP kinase . Stripping and reprobing these membranes for total erk1 levels showed similar expression in all cells tested (data not shown). Therefore, the level of MAP kinase activity depends on the expression of specific integrins and their cytoplasmic domains. We next investigated whether altering the activation state of MAP kinase could reverse the hα5 or hα6A induced phenotypes. We manipulated the level of active MAP kinase through overexpression of MEK-1, an upstream activator of MAP kinase, or by addition of PD-98059, a specific MEK inhibitor . Cotransfection of myoblasts with constitutively active MEK and hα6A integrin restores a proliferative phenotype to myoblasts expressing hα6A. These cells stably express both the hα6A subunit and CA-MEK after drug selection and continue to proliferate for the lifetime of the cells in culture. FACS analysis of propidium iodide labeled cells shows an increased ratio of G2 to G1 cells in the hα6A/ CA-MEK cotransfectants (data not shown). As reported previously , we were unable to isolate cells stably overexpressing only the α6A integrin. The hα6A-CA-MEK transfected cells are similar to the hα5 transfected myoblasts; i.e., they remain proliferative and do not differentiate appreciably compared with hα6A transfected or UT cells . The level of active MAP kinase is enhanced in hα6A-CA-MEK cells when compared to hα6A or UT myoblasts. Interestingly, we were unable to obtain stable expression of CA-MEK in untransfected, control myoblasts. Presumably, excessive levels of activated MEK, or MAP kinase, leads to increased cell death or decreased cell growth. We next determined whether decreasing the level of active MAP kinase, using the specific MEK inhibitor PD-98059, would reverse the hα5 phenotype. hα5 transfected myoblasts were plated onto FN-coated plates in serum-containing medium and allowed to attach for 8–12 h. Increasing concentrations of the MEK inhibitor, PD-98059 were then added for an additional 24–48 h. With increasing inhibitor concentration, the fraction of differentiated cells increased, whereas at high inhibitor concentrations, the total number of cells decreased, presumably due to inhibited proliferation . After 48 h, hα5 transfected myoblasts treated with a 25 μM or greater concentrations of the MEK inhibitor displayed marked differentiation into myotubes compared with untreated hα5 transfected cells resembling UT controls . A Western blot for the level of active MAP kinase shows that increasing concentrations of PD-98059 reduces MAP kinase activity in hα5 transfected cells . Taken together, our observations demonstrate that quantitative changes in integrins closely parallels changes in MAP kinase activation. Moreover, the level of active MAP kinase appears to be a critical determinant of myoblast proliferation versus differentiation. In this study we addressed the mechanisms by which integrin α subunits modulate intracellular signal transduction events that lead to phenotypic changes in cell proliferation and differentiation. Skeletal myoblasts are well suited for this kind of study. The probability that a myoblast withdraws from the cell cycle and initiates terminal differentiation is highly sensitive to environmental cues including growth factors and ECM components . The sharply contrasting effects of ectopic α5 or α6A integrin on myoblast proliferation and differentiation therefore provide a useful system to address α subunit specific signaling and the mechanisms by which integrins regulate the myoblast decision to withdraw from the cell cycle and initiate terminal differentiation. The data presented here demonstrate that the ectopic α subunits differentially regulate proliferative signaling through the β1 subunit. They also demonstrate that paxillin, FAK, and MAP kinase serve as important regulators of cell cycle withdrawal and the onset of terminal differentiation. Finally, our results show that the decision to withdraw from the cell cycle and initiate terminal differentiation is highly poised and regulated by quantitative changes in the level of MAP kinase activation, which in turn is regulated by quantitative changes in levels of integrin signaling. These observations provide a rationale for: (a) the apparently disparate requirements of different muscle cell lines and primary cultures for proliferation and cell cycle withdrawal, (b) the modulation of integrin subunits during muscle differentiation and (c) the hyperproliferative phenotypes observed when integrins are modulated in some other cell types . How does the α5β1 integrin potentiate signaling while the α6β1 integrin attenuates it? Our data show that the β1A subunit (i.e., cytoplasmic domain) is sufficient to transmit proliferative signals. The α5 subunit is permissive and allows β1 signaling while the α6A inhibits it. Previous studies indicate that signaling through integrins requires receptor ligation or clustering into focal adhesions . In the myoblast system, both the ectopic α5 and α6A subunits form functional receptors with the endogenous β1 subunit for FN or LM, respectively . Therefore, the differences in signaling we observe are most likely not due to an inability to bind ligand. Furthermore, since IL2R-β1A alone stimulates proliferation, receptor-ligation does not appear necessary except perhaps to form focal adhesions. Our observation used cells cultured on a complex ECM containing serum and secreted FN, also indicating that receptor ligation may not be a critical factor, for the inhibitory effects of ectopic α6. Interestingly, the α5β1 and the α6β1 integrins exhibit distinct subcellular distributions in muscle when cultured on the appropriate matrix ligand (Sastry, S., J. Muschler, and M. Lakonishok, unpublished observations). The α5β1 integrin localizes in focal adhesions on a FN substrate. In contrast, the α6β1 integrin displays a diffuse cell surface distribution on a laminin substrate. Since the initiating event for signaling through integrins is receptor clustering, the contrasting localizations of α5β1 and α6β1 could reflect a difference in their ability to signal. The clustering of integrins recruits signaling proteins into the focal adhesion signaling complex . In this view, the α5β1 integrin (or any other β1 integrin with a permissive α subunit) recruits and activates proteins like FAK, paxillin, and MAP kinase to stimulate signaling. The α6β1 integrin, though able to bind to LM, is unable to recruit and/or activate the requisite signaling complex. In the context of a model in which integrin signaling occurs primarily through the β1 subunit, how might the α subunits regulate signaling? A possible mechanism is that the α5 and α6A cytoplasmic domains differentially regulate the accessibility of binding sites on the β1 cytoplasmic domain. The α5 subunit (and likely several other α subunits) would permit exposure of critical binding sites, whereas the α6A subunit would mask them. A similar mode of regulation is proposed for the α subunit cytoplasmic domain in the ligand-dependent localization of integrins in focal adhesions . Other models of regulation are also possible. They include a steric masking of the β1 cytoplasmic domain by the α6 cytoplasmic domain through interaction with an inhibitory binding protein, or through an α6-mediated signaling event that results in the activation of a phosphatase. The observation that IL2R-β1A expression rescues the hα6A phenotype, suggests that α6A masks some critical site on β1A cytoplasmic domain. Our observation that ectopic IL2R-α6A does not inhibit proliferation argues against an α6β1 binding protein but does not rule it out. The critical regulatory site(s) within the α6 cytoplasmic domain appears to reside in the eleven COOH-terminal amino acids of the α6A sequence (Table I ). This COOH-terminal region of the α6A cytoplasmic domain contains two putative serine phosphorylation sites , which could potentially participate in negative regulation of signaling by the α6A integrin. Preliminary mapping of the α6A cytoplasmic domain implicates one of these phosphorylation sites in α6A in inhibition of proliferation (unpublished observations). However, the physiological relevance of α6 phosphorylation in this system is not yet clear. Our observations also identify important roles for intracellular components of integrin signaling pathways in regulating cell cycle withdrawal and the onset of terminal differentiation. FAK and paxillin, stand out as potential integrin proximal mediators of adhesive signaling. These proteins are phosphorylated on tyrosine in response to cell adhesion to ECM in many cell types . In addition, both FAK and paxillin bind to synthetic peptides derived from the β1A cytoplasmic domain . However, their role in cell cycle withdrawal and the onset of terminal differentiation is not well understood. Some evidence implicates FAK in proliferation and cell survival. Displacement of endogenous FAK from focal adhesions in endothelial cells, through microinjection of the focal adhesion targeting domain, interferes with cell cycle progression resulting in apoptosis. Similarly, microinjection of β1A peptides corresponding to the FAK binding site, or anti-FAK antibodies induces apoptosis in cultured fibroblasts . Expression of constitutively active FAK, CD2-FAK, in epithelial cells protects them from apoptosis . Our data support a requirement for FAK activation in basal myoblast proliferation; but it does not contribute to the enhanced proliferation, i.e., the inhibited cell cycle withdrawal, observed in cells expressing ectopic α5. Phosphorylation of FAK on tyrosine is not significantly altered in myoblasts expressing ectopic α5 integrin. In most cell types, FAK phosphorylation peaks within 1 h after cell attachment to the ECM . A transient increase in FAK phosphorylation is observed in α5 transfected myoblasts after initial attachment on a FN substrate (data not shown). However, we do not observe a sustained increase in FAK activation that coincides with the sustained proliferative phenotype induced by ectopic α5 integrin. Additionally, overexpression of FAK, as well as several FAK mutants and FRNK , had no detectable effect on myoblast proliferation or differentiation (unpublished observations). However, ectopic expression of CD2-FAK, a membrane-bound, activated form of FAK, does promote proliferation and inhibit differentiation, thus resembling the effect of ectopic α5 or β1A integrin. Presumably this arises because CD2-FAK is targeted to the membrane and therefore available to recruit additional adhesive signaling complexes. In contrast, the level of FAK phosphorylation is reduced in myoblasts expressing ectopic α6A integrin, correlating with the increased cell cycle withdrawal. Ectopic expression of CD2-FAKY397F, which lacks an autophosphorylation site, acts as a dominant negative and inhibits proliferation and promotes differentiation, thus revealing its role in basal proliferation. This phenotype is similar to but less dramatic than that of ectopic α6A integrin expression. It is interesting to note that Y397 is a binding site for src family kinases . Finally, coexpression of CD2-FAK and hα6A results in proliferation. Taken together, these observations are consistent with a role for FAK phosphorylation in the basal level of proliferation seen in control myoblasts and show that it lies on the pathway downstream of the α6A-mediated inhibition of the β1 integrin signaling. In contrast to FAK, both the expression and subsequent tyrosine phosphorylation of paxillin are significantly enhanced in α5 transfected myoblasts when compared to untransfected or α6A transfected cells. A role for paxillin in proliferation or differentiation has not been demonstrated previously. Paxillin is a multidomain adapter protein that mediates numerous interactions with different signaling proteins, including FAK . However, paxillin has no known enzymatic activity. Paxillin does contain multiple LIM domains, which are involved in protein-protein interactions and targeting of paxillin to focal adhesions . Finally, paxillin has been implicated in growth factor dependent differentiation where an increase in its tyrosine phosphorylation correlates with neuronal differentiation . In myoblasts, the upregulation of paxillin in response to ectopic α5 integrin coincides with a proliferative phenotype, which can be recapitulated by ectopic expression of paxillin. Our results with paxillin mutants implicate both the Y118F and S188/190A phosphorylation sites as key. Whereas the mechanism by which paxillin inhibits cell cycle withdrawal, i.e., stimulates proliferation, is not known, increased paxillin expression and phosphorylation enhances the formation of adhesive signaling complexes as do CD2-FAK and ectopic α5 expression (unpublished observation). In contrast to the significant effects of ectopic α5 expression on paxillin expression and phosphorylation, ectopic α6A expression had only a very modest effect on paxillin phosphorylation and none on expression. Coexpression of paxillin and hα6A did not relieve the α6A phenotype (data not shown). Thus the level of paxillin expression and phosphorylation appear to contribute the myoblast decision to proliferate or differentiate. However, it may not play a role in the inhibition of proliferation by ectopic α6A expression. It is noteworthy that our results also indicate an uncoupling of FAK and paxillin signaling, since they are not activated in parallel as reported for fibroblasts . Although our results demonstrate an inhibitory role for the α6A integrin in signaling, this integrin is capable of activating intracellular signals in other cell systems. In macrophages, for example, α6Aβ1-mediated attachment to laminin leads to increased tyrosine phosphorylation of paxillin . In addition, antibody clustering of the α6 integrin in endothelial cells leads to a distinct profile of tyrosine phosphorylated proteins, which do not include FAK or paxillin . The contrasting effects of ectopic α5 or α6A integrin expression on MAP kinase activation suggests quantitative changes in MAP kinase activation plays a major role in regulating myoblast cell cycle withdrawal. Perturbing the ratio of different integrins in the cell alters the level of MAP kinase activation. In contrast to our findings, the α6A integrin, but not the α6B subunit, activates MAP kinase in macrophages . Cell type differences likely explain these conflicting observations. In the muscle system, the effect of ectopic α5 or α6A integrins and their mutants on MAP kinase activation parallels their phenotypic effects on myoblast proliferation and differentiation. Likewise, perturbing the activation state of MAP kinase, using a MEK inhibitor or CA-MEK, can overcome the integrin induced effect on both MAP kinase activity and the myoblast phenotype. Consistent with these results, the activation of MAP kinase is apparently an important determinant of myogenic differentiation. Inactivation of MAP kinase by overexpression of MAP kinase phosphatase renders C2C12 myoblasts insensitive to mitogenic stimuli, favoring expression of muscle-specific genes like myoD . Finally, our data show that MAP kinase activation in myoblasts requires both adhesive and growth factor signals. We do not observe active MAP kinase in the absence of serum or on a non-specific adhesive substrate like poly- l -lysine (unpublished observations). Taken together, these findings reveal a role for integrins in controlling myoblast proliferation and differentiation through a pathway that regulates the level of MAP kinase activation. The ability of different integrins to activate MAP kinase is proposed to occur via an association of integrins with the adaptor protein, shc. In fibroblasts or endothelial cells, α5β1 and several other integrins interact with shc and thus can activate MAP kinase whereas the α6 integrin cannot bind shc or activate MAP kinase . However, the interaction with shc does not require integrin cytoplasmic domains. Our data do not preclude an involvement of shc; the increase in MAP kinase activation seen in response to the α61044t truncation may reflect a difference in the binding of shc, or an unidentified adapter or regulatory protein. Our observations can be summarized in a working model for adhesive regulation of myoblast withdrawal from the cell cycle and the onset of terminal differentiation. In this model, the myoblast decision to proliferate or withdraw from the cell cycle is regulated, at least in part, by the level of activated MAP kinase. This decision appears highly poised and sensitive to quantitative fluctuations in the level MAP kinase activation: increased MAP kinase activation favoring proliferation and decreased MAP kinase activation favoring cell cycle withdrawal. MAP kinase activation, in turn, is regulated by the signaling emanating from both adhesive and growth factor pathways. Evidently, neither pathway alone is sufficient to sustain proliferation. The absence of either growth factor or adhesive signaling promotes cell cycle withdrawal and the onset of terminal differentiation, which appears to function as a default. At one level, the synergy is compatible with an additive model since increased signaling via either enhanced growth factor or adhesive signaling leads to a decreased probability of cell cycle withdrawal. However, the synergy between the growth factor and adhesive signaling systems likely has interactions that are more consistent with an additive, threshold model, in which signals are required from both the growth factor and adhesive pathways. MAP kinase is not activated and targetted to the nucleus in cells that are either adhering to a nonspecific adhesive substrate like poly- l -lysine or that lack serum growth factors. Recent studies point to RAF as an integration point for adhesive and growth factor signals . This working model provides a rationale for diverse observations on the myoblast decision to proliferate or differentiate. Infection of myoblasts with viruses encoding src family kinases or erbB , a truncated form of the EGF receptor with intrinsic tyrosine kinase activity, or the ectopic expression of CD2-FAK or paxillin all inhibit cell cycle withdrawal and produce a proliferative phenotype similar to that of myoblasts ectopically expressing the α5 integrin. It is likely that this arises from increased adhesive and/or growth factor signaling resulting in increased MAP kinase activation. Similarly, the general requirement for high confluency and low serum for differentiation of diverse muscle cell lines likely also appears to reflect enhanced mitogenic signaling, i.e., increased MAP kinase activation, which in this case results from immortalization or adaptation to culture. Previous reports show alterations in the expression of integrin subunits during muscle differentiation, our results raises the possibility that they contribute to cell cycle withdrawal and the onset of terminal differentiation. Finally, our studies appear pertinent to keratinocyte proliferation in vivo as well as to myoblast differentiation in vitro. Transgenic mice expressing two- to threefold increased expression of α5β1 or α2β1 integrins exhibit epidermal hyper-proliferation, perturbed keratinocyte differentiation and other features of psoriasis, a skin disease . This epidermal hyper-proliferation is similar to our observations on myoblasts expressing ectopic α5 integrin. Our working model provides a possible mechanism. Therefore, it is likely that quantitative changes in mitogenic signaling through alterations in adhesive signaling produces phenotypic alterations that operate through common mechanisms in diverse systems. | Study | biomedical | en | 0.999996 |
10087272 | Mouse L cells were grown in DME supplemented with 10% FCS. Transfectants expressing E-cadherin , nEα(1-906), nEαN(1-508), and nEαC(509-906) , and other E-cadherin–α-catenin fusion molecules were grown in the same medium containing 150 μg/ ml of G418. nEα(1-906), nEαN(1-508), and nEαC(509-906) were originally called nEα, nEαN, and nEαC, respectively . We named transfectants by combining the name of the fusion protein with L; for example, L cells expressing nEα(1-906) were designated as nEα(1-906)L cells. Mouse PC9 cells were grown in a 1:1 mixture of DME and Ham's F12 supplemented with 10% FCS (DH10). PC9 cells expressing α(1-184/509-643)-HA (see below) were grown in the same medium containing 150 μg/ml of G418. Human colon carcinoma DLD-1/R2/7, abbreviated to R2/7, and its transfectants expressing αE(1-890), αE(1-325/510-890), αE(1-509), and αE(1-325) were also cultured in DH10 medium. Mouse anti–ZO-1 mAb (T8-754) was obtained and characterized as described . Anti–E-cadherin mAb (ECCD-2), which was concentrated by ammonium sulfate precipitation, was a generous gift from Dr. M. Takeichi . Mouse anti-vinculin mAb (hVIN-1) and mouse anti–α-actinin mAb (BM-75.2) were purchased from Sigma Chemical Co. Mouse anti–HA-tag mAb (12CA5) was purchased from Boehringer Mannheim Biochemicals . Cy2-labeled anti–rat IgG and Cy3-labeled anti–mouse IgG antibodies were purchased from Amersham . DTAF (dichlorotriazinyl amino fluorescence)-labeled anti–rat IgG was purchased from Chemicon International, Inc. We constructed pBATEα(327-906), pBATEα(631-906), pBATEα(1-643), pBATEα(509-643), pBATEα(1-325/509-906), and pBATEα(1-402/509-906), expression vectors for nEα(327-906), nEα(631-906), nEα(1-643), nEα(509-643), nEα(1-325/509-906), and nEα(1-402/509-906), respectively . For construction of these vectors, we used three plasmids: (a) pBATEM2, mouse E-cadherin expression vector , the ClaI-XbaI fragment of which corresponds to the catenin-binding site and was replaced with the α-catenin cDNA fragments in constructs; (b) pSK102B, which contains the α-catenin cDNA with a PstI-BglII adaptor inserted into the PstI site just before the initiation methionine codon; and (c) pBATEα, the ClaI-XbaI fragment of pBATEM2 was replaced with the BglII-XbaI fragment of pSK102B including the whole ORF of the α-catenin cDNA . In the open reading frame of the α-catenin cDNA sequence, we used four restriction sites, PmaCI, ScaI, ClaI, and SmaI, corresponding to amino acid residues 326, 403, 508, and 670, respectively. The XbaI site at the 3′ terminal of α-catenin cDNA in pSK102B was also used. For the production of pBATEα(327-906), the ClaI-XbaI fragment of pBATEM2 was replaced with the PmaCI-XbaI fragment of pSK102B. For construction of pBATEα(631-906), a ClaI site (630ClaI) was introduced at the position corresponding to amino acid residue 630 of α-catenin cDNA in pSK102B by PCR, then the ClaI-XbaI fragment of pBATEM2 was replaced with the 630ClaI-XbaI fragment. For production of pBATEα(1-643), an XbaI site (644XbaI) was introduced at a position corresponding to amino acid residue 644 of α-catenin cDNA in pSK102B by PCR, then the ClaI-XbaI fragment of pBATEα was replaced with the ClaI-644XbaI fragment. For production of pBATEα(1-325/509-906) and pBATEα(1-402/509-906), the PmaCI-ClaI and the ScaI-ClaI fragments, respectively, were excised from pBATEα. All junctions newly produced in the α-catenin coding sequence were arranged in frame using adaptors or linkers as necessary. For construction of pEFα(1-184/509-643)-HA, an EcoRI site (185 EcoRI) was introduced at the position corresponding to amino acid residue 185 of α-catenin cDNA in pSK102B by PCR. The BglII-185EcoRI fragment, ClaI-644XbaI fragment, and a HAα3′ fragment, which contains an HA epitope tag sequence, a stop codon, and 3′ noncoding region of α-catenin, were tandemly ligated and inserted into the pEFMC1-neo expression vector . We also constructed pGEX-αN(1-508), pGEX-αC(509-906), and pGEX-α(671-906), expression vectors for GST–α-catenin fusion molecules, using pSK102B and pGEX vectors ( Pharmacia LKB Biotechnology ). For production of pGEX-αN(1-508), pGEX-αC(509-906), and pGEX-α(631-906), the BglII-ClaI, ClaI-XbaI, and SmaI-EcoRI fragments of pSK102B were inserted into the BamHI-SmaI sites of pGEX-2T, the SmaI site of pGEX-3X, and the SmaI-XhoI sites of pGEX-4T-3 ( Pharmacia LKB Biotechnology ), respectively. L cells (5 × 10 5 per 3-cm plate) were cotransfected with 1 μg of each expression vector and 0.05 μg of pSTneoB by the lipofectamine method (Life Technologies, Inc.). After 48 h of incubation, the cells were replated on pairs of 9-cm dishes and cultured in the presence of 400 μg/ml G418 to select stable transfectants. Colonies of G418-resistant cells were isolated, recloned, and subsequently maintained in complete medium with 150 μg/ml of G418. We isolated several stable clones for each transfection experiment. Since nEα(327-906)L-11, nEα(631-906)L-7, nEα(1-643)L-9, nEα(509-643)L-32, nEα(1-325/509-906)L-2, and nEα(1-402/509-906)L-23 clones expressed relatively large amounts of fusion molecules, we mainly used these in this study. Trypsinized PC9 cells (10 5 ) were suspended in 500 μl of Hepes-buffered (pH 7.4) Ca 2+ - and Mg 2+ -free saline and mixed with 10 μg of expression vector and 1 μg of pSTneoB. Electroporation was performed at 960 μF, 250 V. The cells were selected in G418 (0.2 mg/ml)-containing medium. All procedures were performed at room temperature. Cells cultured on coverslips were fixed with 3.5% (for hVIN-1 and BM-75.2) or 1.0% (for T8-754) formaldehyde solution in HMF (Hepes-buffered magnesium-free saline) for 15 min. After three washes with PBS, cells were soaked in blocking solution (1% BSA in PBS) for 30 min and subsequently incubated with ECCD-2 diluted with PBS containing 1% BSA for 30–60 min at room temperature. The cells were then washed three times with PBS and soaked in 0.2% Triton X-100 in PBS for 15 min. After rinsing with PBS, the cells were treated with 1% BSA in PBS for 30 min and subsequently incubated with hVIN-1, T8-754, or BM-75.2 for 30–60 min. After extensive washing with PBS, the specimens were incubated with fluorescence-labeled second antibodies (Cy2- or DTAF-labeled goat anti–rat IgG for ECCD-2 and Cy3-labeled donkey anti–mouse IgG [H&L] for hVIN-1, T8-754, and BM-75.2) diluted with PBS containing 1% BSA for 30 min at room temperature. After washing thoroughly with PBS, the preparation was mounted with 90% glycerol-PBS containing 0.1% para-phenylendiamine and 1% n -propylgalate. Samples were observed with a Zeiss Axiophot photomicroscope ( Carl Zeiss ). Images were recorded with a cooled CCD camera controlled by a Power Macintosh 7600/132 and the software package IPLab Spectrum V3.1 (Signal Analytic Corp.). SDS-PAGE (10 or 7.5%) and immunoblotting were performed as described previously . Samples were solubilized in SDS sample buffer, separated by SDS-PAGE, and gels were stained with Coomassie brilliant blue R-250. For immunoblotting, proteins were electrophoretically transferred onto nitrocellulose sheets. Nitrocellulose membranes were then incubated with ECCD-2, T8-754, or 12CA5. Antibody detection was performed using an Amersham biotin-streptavidin kit with biotinylated anti–rat or anti–mouse Ig and NBT-BCIP. In vitro binding assays were performed as previously described . In brief, GST–α-catenin fusion proteins were expressed in Escherichia coli and purified using glutathione-Sepharose 4B beads ( Pharmacia LKB Biotechnology ) as previously described . Then, 2 ml of the cell lysate of Sf9 cells expressing N-ZO-1 was added, followed by incubation for 3 h at 4°C. The beads were again washed with PBS containing 0.1% Triton X-100, 2 mM PMSF, and 4 μg/ml of leupeptin, and then bound proteins were eluted with 1 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 10 mM glutathione. The amounts of GST fusion proteins in each eluate were determined by SDS-PAGE. Confluent cultured cells (∼4 × 10 6 cells per 6-cm dish) were extracted with 2.5% NP-40 in HMF, then centrifuged at 100,000 rpm for 30 min as described previously . To the supernatant, 2× SDS sample buffer was added to make the total volume 0.4 ml and used as the detergent-soluble fraction. On the other hand, the pellet fraction was dissolved in 0.4 ml of 1× SDS sample buffer and used as the detergent-insoluble fraction. Cells were trypsinized by two different methods for the differential removal of E-cadherin or its fusion molecules, as described by Takeichi . In brief, cells were treated with 0.01% trypsin in the presence of 1 mM CaCl 2 (TC treatment) or 1 mM EGTA (TE treatment) at 37°C for 30 min. Generally, cadherins are left intact after TC treatment, but are digested by TE treatment. For the cell aggregation assay, cells were dispersed after TC treatment as described by Takeichi . L and R2/7 transfectants were pretreated with 1 μM cytochalasin D in culture medium for 2 h. Although this treatment is necessary for the dissociation of L cells expressing nEαC(509-906) and R2/7 cells expressing αE(1-890), it did not affect cell aggregation of other transfectants. Aliquots of 5 × 10 5 dissociated cells were plated in each well of a Falcon 12-well plate with 0.5 ml HMF and allowed to aggregate, as described previously . The extent of cell aggregation was represented by the index N t / N 0 where N t is the total particle number after incubation time t and N 0 is the total particle number at the initiation of incubation. For the cell dissociation assay, confluent cultures were treated with TC and TE and dissociated by pipetting 10 times . The extent of cell dissociation was represented by N TC / N TE , where N TC and N TE are the total particle number after TC and TE treatment, respectively. In some experiments, confluent cultures were pretreated with 1 μM cytochalasin D in culture medium for 2 h, then cell dissociation analyses were performed in the presence of 1 μM cytochalasin D. In L cell transfectants expressing E-cadherin, two cytoskeletal proteins, vinculin and ZO-1, are precisely colocalized with E-cadherin at cell–cell contact sites . In parental L cells, vinculin is concentrated exclusively at cell-substrate AJ, and ZO-1 does not show specialized localization but some condensation at tips of cellular processes (data not shown). To determine whether α-catenin is involved in the recruitment of vinculin and ZO-1 to E-cadherin–based cell adhesion sites, we used L cell transfectants expressing nEα(1-906), which is a fusion molecule consisting of nonfunctional E-cadherin lacking its catenin-binding domain and full-length α-catenin . As previously reported, this molecule showed similar cell adhesion and cytoskeleton interaction activities to the normal E-cadherin–catenin complex. Immunocytochemical analysis clearly revealed that both vinculin and ZO-1 were precisely colocalized with nEα(1-906) at cell–cell contact sites in L cell transfectants . As reported previously, nonfunctional E-cadherin does not interact with the cytoskeleton and nEα(1-906) is not associated with endogenous β-catenin . These observations indicated that α-catenin was crucial for the recruitment of vinculin and ZO-1 to cell–cell contact sites in transfected L cells. To determine the domain of α-catenin necessary for the recruitment of vinculin or ZO-1, we constructed several expression vectors encoding various E-cadherin–α-catenin fusion molecules, in which distinct domains of α-catenin were deleted . These expression vectors were introduced into mouse L cells, and stable transfectant clones were isolated for each construct. Each mutant molecule expressed in transfectants had the expected apparent molecular mass . The subcellular localization of vinculin was compared with those of expressed fusion molecules in various transfectants . nEα(327-906) in which the NH 2 -terminal 326 residues had been truncated was precisely colocalized with vinculin . However, nEαC(509-906) with a longer NH 2 -terminal deletion showed no colocalization with vinculin . In cells expressing nEαN(1-508) with truncation of the COOH-terminal 398 residues, expressed fusion molecule was also colocalized with vinculin at sites where it was heavily condensed (data not shown). These observations suggested that residues 327-508 are important for the recruitment of vinculin. Consistently, vinculin was not colocalized with nEα(1-325/ 509-906) lacking residues 326-508 . Residues 326-508 of α-catenin include the direct α-actinin-binding site (325-394 residues) reported previously . When this α-actinin-binding site was added to nEα(1-325/509-906), the resultant fusion molecule nEα(1-402/509-906) retained the ability to colocalize with vinculin . Double-immunostaining for E-cadherin and α-actinin revealed that the constructs which recruited vinculin, such as nEα(327-906) and nEα(1-402/509-906), could recruit α-actinin . In contrast, nEα(1-325/509-906) was not colocalized with either vinculin or α-actinin . These results demonstrated that residues 327-402 of α-catenin are crucial for the recruitment of vinculin as well as α-actinin to cadherin-based cell adhesion sites of L cell transfectants . To determine the domain(s) of α-catenin involved in the recruitment of ZO-1, we compared the subcellular localization of ZO-1 with that of expressed fusion molecules in transfectants. In contrast with vinculin, ZO-1 was not precisely colocalized with nEαN(1-508) but with nEαC(509-906) . nEα(631-906), which had the longest NH 2 -terminal deletion and showed no cell adhesion activity (see below), was also colocalized with ZO-1 at cell–cell boundaries , although their condensation at cell–cell boundaries was not as exclusive compared with those in other transfectants. These observations strongly suggested that the COOH-terminal domain of α-catenin (631-906 residues) is crucial for the recruitment of ZO-1 . We have reported previously that the NH 2 -terminal half of ZO-1 (N-ZO-1) directly interacts with α-catenin . Therefore, it was expected that N-ZO-1 would directly interact with the COOH-terminal domain of α-catenin. To test this possibility, we produced four GST fusion proteins, GST-α(1-906), GST-αN(1-508), GST- αC(509-906), and GST-α(671-906), which contained the full-length α-catenin, its NH 2 -terminal half, its COOH-terminal half, or the COOH-terminal 236 residues, respectively . Then, we analyzed in vitro binding abilities of these fusion molecules with recombinant N-ZO-1 produced in Sf9 cells by baculovirus infection. As shown in Fig. 6 B, N-ZO-1 bound to not only GST-α(1-906) but also GST-αC(509-906) and GST-α(671-906), although the binding affinities to the latter two were lower than that to the former. N-ZO-1 did not bind to GST-αN(1-508). These results strongly suggested that the COOH-terminal 276 amino acids (residues 631-906) of α-catenin recruited ZO-1 to cadherin-based cell–cell contact sites through its direct binding to ZO-1 in transfected L cells. We have reported previously that nEαC(509-906) shows similar cell adhesion activity to the normal E-cadherin– catenin complex . To determine the domain which is required for this function, we compared the reaggregative properties of L cell transfectants expressing nEα(509-643) and nEα(631-906); the former was an nEαC(509-906) derivative lacking the ZO-1-binding domain, and the latter contained only the ZO-1-binding domain . Cells expressing nEα(509-643) aggregated as rapidly as those expressing nEαC(509-906). In contrast, the reaggregation of cells expressing nEα(631-906) was indistinguishable from that of parent L cells . These observations suggested that residues 509-643 of α-catenin are required for cell adhesion activity of fusion molecules. Indeed, when residues 509-643 were added to nEαN(1-508), cells expressing the resultant fusion molecule nEα(1-643) aggregated rapidly, although nEαN(1-508) did not . The levels of expression of nEα(631-906) or nEαN(1-508) in the transfectants examined were relatively low among the fusion molecules examined . However, the reduced level of fusion molecule expression did not seem to be the cause of loss of their cell adhesion activity since L cells expressing similar or lesser amounts of nEα(1-643) still aggregated rapidly (data not shown). These results demonstrated that residues 509-643 of α-catenin are required for cell adhesion activity of E-cadherin–α-catenin fusion molecules in transfected L cells . We tentatively called this an adhesion-modulation domain . The cell adhesion activities of E-cadherin and its α-catenin fusion molecules were reported to be associated with their interactions with the cytoskeleton . For example, about half of the nEαC(509-906) was resistant to extraction with NP-40 Interestingly, most of the nEα(509-643) carrying only the adhesion-modulation domain was extracted with NP-40, suggesting that this fusion molecule did not interact with the cytoskeleton . In contrast, nEαN(1-508) and nEα(631-906) did not show cell adhesion activity but interacted with the cytoskeleton as judged from their resistance to NP-40 extraction. In the cell aggregation assay, the disorganization of actin-based cytoskeleton by cytochalasin D treatment essentially did not affect the cell aggregating activity of any of the transfectants (data not shown). These results suggested that cadherin-based cell adhesion activity and cadherin–cytoskeleton interaction are regulated independently by distinct domains of α-catenin. We demonstrated previously that cells assumed two states of cadherin-based cell adhesion, strong and weak . The cell–cell adhesion in the strong state could hardly be dissociated by pipetting, although that in the weak state was easily dissociated. Using the cell dissociation assay, we examined the states of cell adhesion of various L cell transfectants. As reported previously, L cell transfectants expressing nEαC(509-906), which contained both the adhesion-modulation and ZO-1-binding domains of α-catenin, were hardly dissociated in the cell dissociation assay, indicating that these cells showed the strong state of cell adhesion activity . Intact E-cadherin or other fusion molecules containing both of these domains also showed the strong state of cell adhesion activity . In contrast, cells expressing nEα(509-643), which contained only the adhesion-modulation domain of α-catenin, were easily dissociated into single cells under the conditions of the cell dissociation assay , indicating that these cells showed the weak state of cell adhesion activity. Interestingly, cells expressing nEα(1-643) lacking only the ZO-1-binding domain also showed the weak state of cell adhesion activity . It should be noted that nEα(1-643) was hardly extracted with NP-40 , suggesting its interaction with the cytoskeleton probably through the vinculin/α-actinin-binding domain. In the presence of cytochalasin D, nEαC(509-906)L cells were also easily dissociated under the same condition , although the amount of expressed nEαC(509-906) was not altered in the presence or absence of cytochalasin D (data not shown). These results indicated that the ZO-1-binding domain, but not the vinculin/α-actinin-binding domain, in addition to the adhesion-modulation domain was required for the strong state of cell adhesion activity. It was also suggested that the intact actin-based cytoskeleton is required for this type of adhesion activity. We examined the role of α-catenin in epithelial cell adhesion using α-catenin–deficient epithelial cell lines and their transfectants expressing α-catenin deletion mutants. Since cadherin–catenin complex is not colocalized with ZO-1 in epithelial cells, the roles of α-catenin in epithelial cells are expected to be different, at least in some aspects, from those in nonepithelial cells such as L cells. DLD-1/R2/7, abbreviated to R2/7, is an α-catenin–deficient colon carcinoma line. R2/7 transfectants expressing α-catenin deletion mutants were previously reported . We used four of these transfectants in this study. α-Catenin deletion mutants expressed in these transfectants are shown in Fig. 9 A. Using the cell aggregation assay, we compared the reaggregative properties of R2/7 and its transfectants . R2/7 itself showed aggregation activity which was blocked in the presence of E-cadherin blocking antibodies . R2/7 transfectants expressing αE(1-890), which is indistinguishable from cells expressing intact α-catenin, aggregated more rapidly than parental R2/7 cells. Not only cells expressing αE(1-325/510-890) but also those expressing αE(1-509), which lacks the adhesion-modulation domain, also aggregated more rapidly than parental R2/7 cells. R2/7 cells expressing αE(1-325) showed similar aggregation activity to the parental cell line R2/7. These results demonstrated that the residues 325-509 including the vinculin/α-actinin-binding domain are also involved in cadherin-dependent cell aggregation activity in R2/7. In the cell dissociation assay, R2/7 was readily dissociated but R2/7 transfectants expressing αE(1-890) were hardly dissociated . Although cells expressing αE(1-325/ 510-890) showed some degree of resistance to pipetting, the strong state of cell adhesion activity was not fully restored . This partial restoration of the strong state of cell adhesion activity was also observed in cells expressing not only αE(1-509) but also αE(1-325) . These results suggested that multiple domains of α-catenin were required for the strong state of cell adhesion activity in R2/7. To confirm the importance of adhesion-modulation domain in epithelial cells, we used PC9 cells, a human lung carcinoma cell line lacking α-catenin expression. It was reported that PC9 showed aggregation activity to some extent and that this activity was dependent on E-cadherin– β-catenin complex without α-catenin . We constructed an expression vector encoding α(1-184/509-643), in which only an adhesion-modulation domain was covalently connected to the NH 2 -terminal β-catenin-binding domain of α-catenin . This vector was introduced into PC9 cells, and several transfectant clones were isolated. α(1-184/509-643) with the expected size was expressed in the transfectants and colocalized with E-cadherin–β-catenin complex (data not shown). Cell aggregation assay revealed that cells expressing α(1-184/509-643) aggregated more rapidly and more extensively than parental PC9 cells . These aggregates were readily dissociated into single cells under the dissociation assay conditions (data not shown). These observations indicated that an adhesion-modulation domain is involved in the weak state of cell adhesion activity even in epithelial cell lines. It is generally accepted that the cadherin–catenin cell adhesion complex plays fundamental roles not only in the formation of cell–cell junctions but also in the morphogenesis of tissue and organs, dependent on its strong state of cell–cell adhesion activity and its interaction with the actin-based cytoskeleton . We identified three distinct functional domains of α-catenin required for the interaction with vinculin, for direct binding to ZO-1, and for the adhesion activity of E-cadherin–α-catenin fusion molecules . Here, we will discuss possible functions of each domain of α-catenin and the relationship between the cell adhesion activity and the interaction of cadherin–catenin complex with the cytoskeleton. We will also discuss the difference of α-catenin function in nonepithelial and in epithelial cells. α-Catenin was reported to be directly associated with β-catenin, α-actinin, and actin filaments, and the domains responsible for their binding have been narrowed down on the α-catenin molecule . It was reported recently that ZO-1 bound to α-catenin directly , but the domain responsible remained elusive. In this study, using deletion constructs, we showed that the COOH-terminal domain (residues 631-906) of α-catenin recruited ZO-1 to the cell adhesion sites and directly bound to NH 2 -terminal half of ZO-1 in vitro. We also demonstrated that this ZO-1-binding domain interacted with cytoskeletons judging from the resistance of fusion molecules to NP-40 extraction. It was reported previously that the COOH-terminal halves of ZO-1 are directly associated with actin filaments . Based on these properties, we can imagine that the ZO-1-binding domain interact with the actin-based cytoskeleton through ZO-1. We demonstrated that this domain is essential for the strong state of cadherin-based cell adhesion in L cell transfectants, which was dependent on the intact actin-based cytoskeleton. The functional importance of the ZO-1-binding domain was also reported using mouse embryos expressing mutant α-catenin . Taken together, we concluded that the ZO-1-binding domain (COOH-terminal 276 residues) of α-catenin plays a fundamental role in the cadherin–catenin cell adhesion system probably through its interaction with ZO-1 and/or the actin-based cytoskeleton. We cannot exclude the possibility that other cytoskeletal proteins interact with the ZO-1-binding domain. This domain is known to interact with actin in vitro . The physiological role of this interaction remains to be elucidated. The ZO-1-binding domain was also shown to directly interact with vinculin in vitro . However, we found that the ZO-1-binding domain in fusion molecules did not recruit vinculin to the cell–cell boundaries in L cell transfectants. Since the reported binding constant of the ZO-1-binding domain to vinculin was lower than that to ZO-1 , the binding of vinculin to the ZO-1-binding domain may be prevented by ZO-1 in L cell transfectants. ZO-2 and ZO-3, homologues of ZO-1, are other candidates as binding proteins to the ZO-1-binding domain. In fact, it was reported recently that ZO-2 showed very similar properties with ZO-1 and directly bound to α-catenin . However, these proteins are not involved in the cadherin-based cell adhesion in L cell transfectants, since their expression was not detected in L cell transfectants (our unpublished observation). We found that residues 327-402 of α-catenin were required for fusion molecules to recruit not only α-actinin but also vinculin in transfected L cells. This is consistent with previous data that this domain directly binds to both vinculin and α-actinin , and these two molecules interact with each other . It is not clear whether vinculin and α-actinin interact with this short domain with 76 residues simultaneously or competitively in vivo. E-cadherin– α-catenin fusion molecules conferred full adhesion activity in L cell transfectants even if they lacked the vinculin/ α-actinin-binding domain. This raised the question of what is the function of the vinculin/α-actinin-binding domain. We reported previously that intact E-cadherin conferred a flexible adhesive phenotype upon L cells, but E-cadherin– α-catenin fusion molecules conferred inflexible phenotypes . If the vinculin/α-actinin-binding domain is involved in this flexible adhesion activity, its function would not be observed using E-cadherin– α-catenin molecules expressed in L cells. On the other hand, it was reported recently that this domain is involved in the organization of apical junctional complex and the activation of cadherin-based cell adhesion in epithelial cells . It has been reported also that vinculin is colocalized with the cadherin–catenin complex in epithelial cells but not in some fibroblastic cell lines , and that vinculin is one of the major components of cell–cell AJ in epithelial cells . These observations suggest that the vinculin/α-actinin-binding domain is required for the function of cadherin–catenin complex, especially for junctional complex formation, only in epithelial cells but not in fibroblastic cells. Deletion constructs showed that residues 509-643 of α-catenin are required for fusion molecules to function as cell adhesion molecules. We tentatively called this domain an adhesion-modulation domain. When mutant α-catenin containing the β-catenin-binding and the adhesion-modulation domains was expressed in α-catenin–deficient PC9 cells, such cells aggregated more rapidly than parental PC9 cells. These results suggested that the adhesion-modulation domain is involved in cell adhesion in the “natural” cadherin/catenin complex. Although several sites of α-catenin were reported to be required for the interaction with the cytoskeleton, the adhesion-modulation domain does not correspond to these cytoskeletal interaction sites. Moreover, the fusion molecule carrying only this domain was easily extracted with NP-40, suggesting that this molecule did not interact with the cytoskeleton. These findings indicated that the adhesion-modulation domain might function without the interaction with the cytoskeleton. It has been accepted that the insoluble fraction of E-cadherin was active in cell adhesion and the soluble one was not, since E-cadherin molecules which could not be extracted with NP-40 were strictly localized at cell–cell contact sites . However, our present results suggested that some fraction of soluble E-cadherin–catenin complex was also active in cell adhesion. The molecular mechanism of the activation of E-cadherin extracellular domain remains unclear. One simple interpretation is that the adhesion-modulation domain supports the lateral aggregation of E-cadherin molecules, which may mediate the weak state of cell adhesion. Alternatively, this domain may trigger off the other adhesion-modulation system. Fusion molecules used contained the membrane proximal, p120-binding domain of E-cadherin and are colocalized with endogenous p120 protein in transfected L cells (our unpublished observation). It was reported that this membrane proximal domain might positively or negatively regulate cadherin-based cell adhesion . It is possible that the adhesion-modulation domain of α-catenin affects the potential activity of the membrane proximal domain of E-cadherin. Some fusion molecules lacking the adhesion-modulation domain were detected at cell–cell boundaries in transfectants, although nonfunctional E-cadherin itself was not . These observations raised the question of how these fusion molecules were condensed at cell–cell boundaries, although they did not function as cell adhesion molecules. The main difference between these fusion molecules and nonfunctional E-cadherin is that the former interacted with cytoskeletal proteins such as ZO-1 or vinculin/α-actinin but the latter did not. As discussed below, ZO-1 is likely to facilitate the lateral aggregation of its membrane binding partners. Vinculin is also expected to form clusters through its interaction with various cytoskeletal components . So, the interaction with cytoskeletal components may cause the clustering of fusion molecules in the plasma membrane, which then induces the association of cadherin complexes on apposed cell membranes . As previously reported, cadherin-based cell adhesion can be classified into the strong state and the weak state, using cell dissociation and aggregation assays . In the cell aggregation assay, cells form aggregates in both adhesive states. In the cell dissociation assay, however, cells in the strong state were hardly dissociated into single cells but those in the weak state were dissociated readily. It is known that the adhesive state is regulated by the phosphorylation level of the cytoplasmic components . We also demonstrated that disorganization of the actin-based cytoskeleton shifted the cadherin-based cell adhesion from the strong to the weak state. Since the level of cadherin expression on the cell surface was not affected in either case, the strong state and the weak state may reflect qualitative differences in cell adhesion activity but not quantitative differences in cell adhesion molecules. The present results are consistent with this idea, since cells in both adhesive states aggregated in a similar manner. We found that all of the fusion molecules that showed the strong state of cell adhesion activity interacted with the cytoskeleton, suggesting that the interaction with the cytoskeleton is required for the strong state of cell adhesion activity. This was supported by the present observation that cytochalasin D treatment shifted cadherin-based cell adhesion to the weak state. Interestingly, the fusion molecule lacking only the ZO-1-binding domain showed the weak state of cell adhesion activity, although it contained a vinculin/α-actinin-binding domain and may interact with the cytoskeletons, judging from the refractoriness to NP-40 extraction. Thus, we concluded that the cytoskeleton interaction through the ZO-1-binding domain, but not through the vinculin/α-actinin binding domain or other domain(s), is required for the strong state of cell adhesion activity in L cell transfectants. As discussed above, ZO-1 and actin are possible binding proteins to this domain in L cell transfectants. ZO-1 is a member of the MAGUK family. Another member of this family, PSD95, is known to facilitate the lateral aggregation of its membrane binding partners such as NMDA receptors and K + channels . Thus, it is possible that ZO-1 strengthens the cell–cell adhesion activity not only by cross-linking α-catenin to actin filaments but also by facilitating the lateral aggregation of E-cadherin or its fusion molecules in L cell transfectants. The role of actin binding to this domain remains unclear. Furthermore, we cannot exclude the possibility that unknown factor(s) interacted with the ZO-1-binding domain and supported the strong state of cell adhesion activity. At the immunoelectron microscopic level, cadherin–catenin complex is known to be colocalized with ZO-1 in nonepithelial cells such as L cells but not in epithelial cells. In epithelial cells, ZO-1 is highly condensed at the TJ and is thought to directly interact with TJ membrane proteins including occludin . These results suggested that the ZO-1-binding domain of α-catenin may have different functions in nonepithelial cells and in epithelial cells. In fact, it was demonstrated that this domain could not cause redistribution of ZO-1 in R2/7, an epithelial colon carcinoma cell line . It remains unclear why the ZO-1-binding domain does not associate with ZO-1 and how this domain functions in epithelial cells. Further studies to address this question will provide important information regarding the mechanisms of junctional complex formation in epithelial cells. It was reported recently that the vinculin/α-actinin-binding domain directly binds to vinculin and this interaction functions to organize the apical junctional complex, including TJ, in R2/7 cells . This domain, instead of the ZO-1-binding domain, functions to recruit ZO-1 at cell–cell boundaries in R2/7 cells. Since we demonstrated that this domain did not interact with ZO-1 directly, the redistribution of ZO-1 may be dependent on the reorganization of TJ caused by α-catenin–vinculin interaction in R2/7 cells. This is consistent with the previous observation that, when the vinculin/α-actinin binding domain was replaced with the vinculin tail domain, the resultant α-catenin–vinculin fusion molecule recruited ZO-1 to cell–cell boundaries . The role of α-catenin in epithelial cell adhesion also seems to be different from that in nonepithelial cells, although the adhesion-modulation domain seems to be generally used in nonepithelial and epithelial cells. We found that the ZO-1-binding domain, which cannot cause redistribution of ZO-1, also failed to fully restore the strong state of cell adhesion activity in epithelial cells, supporting our speculation that ZO-1 is required for the strong state of cell adhesion activity. In epithelial cells, both the NH 2 -terminal half and COOH-terminal half domains are partially involved in the strong state of cell adhesion activity and these two domains cooperatively support this activity. Thus, α-catenin in epithelial cells supports the strong state of cell adhesion activity in a different manner from that in nonepithelial cells. In fact, it was reported recently that the COOH-terminal 236 residues, the in vitro binding domain to ZO-1, were not involved in the cadherin-based cell adhesion activity in epithelial cells, although the COOH- terminal 273 residues of α-catenin were involved in this activity . The 37 residues between 634 and 670 of α-catenin were not involved in the adhesion activity or binding to ZO-1 in L cell transfectants (our unpublished observation). The mechanism by which α-catenin is involved in these processes in epithelial cells remains to be elucidated. Of course, here, we cannot completely exclude the possibility that differences of α-catenin functions observed in L cells and R2/7 cells are not due to differences of the cell type (nonepithelium versus epithelium) but that of the expressed molecule (fusion versus nonfusion). However, our recent observations using α-catenin–deficient F9 cells did not favor this possibility (Maeno, Y., and A. Nagafuchi, unpublished observations). In this study, we identified three functional domains of α-catenin, i.e., the ZO-1-binding domain, vinculin/α-actinin-binding domain and adhesion-modulation domain. We also clarified the effects of the cytoskeleton interaction on the different states of cadherin-based cell adhesion activity. Furthermore, we demonstrated that α-catenin may have different functions in nonepithelial and epithelial cells. Further studies of the regulatory mechanism of cadherin-based cell adhesion by α-catenin will lead to a better understanding of the physiological functions of the cadherin–catenin complex which plays a pivotal role in morphogenesis in multicellular organisms. | Study | biomedical | en | 0.999997 |
10087273 | Monoclonal antibodies against the intracellular (SK15, SK18) and extracellular (BK2, BK9) domains of PTPμ have been described . The MB4 monoclonal antibody was generated against the peptide CSH 338 from the immunoglobulin domain of PTPμ (amino acids 231–256). The 494 polyclonal antibody generated against a PTPμ peptide (amino acids 42–60) has been described . A monoclonal pan-cadherin antibody (SMP) generated against the COOH terminus of chick N-cadherin was obtained from Sigma Chemical Co. The NCD-2 monoclonal antibody was generously provided by Dr. Gerald Grunwald (Thomas Jefferson University, Philadelphia, PA) from hybridoma cells generated by Dr. Masotoshi Takeichi . A polyclonal antibody against N-cadherin was kindly provided by Dr. John Hemperly ( Becton Dickinson Labs, Research Triangle Park, NC). Antibodies against chick NCAM (5e, RO28 and RO32) and L1 (RO21) were generously provided by Dr. Urs Rutishauser (Case Western Reserve University). Monoclonal antibodies against chick L1 (8D9), a bipolar neuron-specific antigen (3G3), and a Müller glia-specific antigen (5A7) were generously provided by Dr. Vance Lemmon (Case Western Reserve University). RPMI 1640 medium, laminin, and fetal bovine serum were obtained from GIBCO BRL . Aprotinin and leupeptin were obtained from Boehringer Mannheim Biochemicals . Tween-20 was obtained from Fisher Scientific Co. All other reagents were obtained from Sigma Chemical Co. Adult rat brains were minced and homogenized (Bellco Biotechnology) in a solution of 0.32 M sucrose in 50 mM Tris-HCl, 150 mM NaCl (TBS), pH 8.0, containing protease inhibitors 5 mM EDTA, 10 μg/ml turkey trypsin inhibitor, 2 mM benzamidine hydrochloride, and 200 μM phenylmethylsulfonylfluoride. The homogenate was layered onto a 0.8 M, 1.2 M sucrose gradient and centrifuged at 25,000 rpm for 45 min (SW28 rotor; Beckman Instruments, Inc. ). The membrane layer was diluted with TBS and respun at 50,000 rpm for 30 min (Ti 70.1 rotor; Beckman Instruments, Inc. ). The pellet was then extracted with 1% sodium deoxycholate in 50 mM Tris-HCl, pH 8.0. The membrane extract was respun at 50,000 rpm for 30 min and the supernatant was incubated overnight at 4°C with CNBr Sepharose 4B beads ( Pharmacia LKB Biotechnology ) that had been covalently coupled with the SK15 PTPμ antibody. The beads were washed extensively with 50 mM Tris-HCl containing 0.5% sodium deoxycholate and 0.5% NP-40, pH 8.0, followed by 10 mM Tris-HCl, pH 8.0. The protein was eluted from the column with 0.1 M diethylamine, pH 11.5, and neutralized with 2 M Tris-HCl, pH 3.6. For SDS-PAGE, sample buffer (4% SDS, 20% glycerol, 0.2 M dithiothreitol, bromphenol blue, 0.12 M Tris, pH 6.8) was added and boiled for 5 min at 95°C. L1 and N-cadherin were purified from chick brains using previously described procedures . Protein purity was checked by two different methods. First, the eluted protein fractions were separated by 4–15% SDS-PAGE (gradient Tris-HCl gels; Bio-Rad Laboratories) and the gel was silver stained. In a second procedure, the eluted fractions were immunoblotted as previously described . 35-mm tissue culture dishes (Falcon Labware) were coated with nitrocellulose in methanol and allowed to dry. A small amount of protein (2–4 μg) was spread across the center of the dishes, and they were incubated 30 min at room temperature. Remaining binding sites on the nitrocellulose were blocked with 2% BSA in PBS, and the dishes were rinsed with RPMI-1640 medium. Retinal explant cultures were made according to a previously described procedure . In brief, embryonic day 8 White Leghorn chick eyes were dissected and the retina was flattened with the photoreceptor side down onto black nitrocellulose filters (0.45-μm pore size; Vanguard International, Inc.) that had previously been incubated in 0.05% concanavalin A to enhance attachment of the retina to the filter. The filter was then cut into 350-μm wide strips perpendicular to the optic fissure using a McIlwain tissue chopper. Strips were inverted onto substrate-coated culture dishes so that the ganglion cell layer was directly adjacent to the substratum. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum/2% chick serum/penicillin-streptomycin-fungizone and incubated at 37°C in 95% air/5% CO 2 . Neurite outgrowth on PTPμ, N-cadherin, and L1 substrates was inhibited by the addition of culture supernatant from the BK2 (anti–PTPμ), NCD2 (anti–N-cadherin), and 8D9 (anti–L1) hybridoma cells, respectively, into the medium at the time of plating retinal explants as described above. The medium was supplemented with fetal bovine serum and chick serum to maintain equivalent levels of growth factors to controls. Neurite outgrowth was examined at 24, 48, and 72 h after plating. Dissociated retina cultures were prepared from E6 embryos using the procedure above, except that the retinas were incubated in 0.25% trypsin and 0.1% EDTA (Mediatech Cellgro) for 20 min at 37°C. The retinas were dissociated by trituration and resuspended in RPMI-1640/10% tryptose phosphate broth/4% fetal bovine serum/1% chick serum/gentamycin, and then cultured in 12-well plates (Falcon Labware). E6 retinas were used because a large percentage of the cells are still mitotic at this age, which is an important requirement for retroviral-mediated gene transfer. Expression of the exogenous gene was repressed by culturing infected cells in the presence of 3 μg/ml tetracycline (see below). For immunohistochemical labeling of retina sections, eyes from E8 chicks were fixed with 4% paraformaldehyde, 0.01% glutaraldehyde in PEM buffer (80 mM Pipes, 5 mM EGTA, 1 mM MgCl 2 , 3% sucrose), pH 7.4. The tissue was rinsed and cryoprotected with 20% sucrose in PBS and frozen in OCT medium (EM Sciences). Cryostat sections were cut at 7-μm intervals, adhered to gelatin coated slides, and stored at −20°C. Sections were permeabilized and blocked with 1% Triton, 20% goat serum in PBS, and then incubated overnight at 4°C with primary antibodies diluted into block buffer (20% goat serum, 1% BSA, 0.5% saponin in PBS). After extensive rinsing, sections were incubated with fluorescein-conjugated secondary antibodies, rinsed extensively with PBS, and then coverslipped with IFF mounting medium (0.5 M Tris-HCl, pH 8.0, containing 20% glycerol and 1 mg/ml p -phenylenediamine). Immunolabeled sections were examined using a 40× objective on an Axiophot microscope ( Carl Zeiss, Inc. ), and images captured using a Hamamatsu-cooled CCD camera. For immunocytochemical labeling of retinal explant cultures, the cultures were fixed as above, and then rinsed with PBS and incubated with block buffer. Cultures were incubated with primary antibodies in block buffer overnight at 4°C. After rinsing with PBS, the cultures were blocked with TNB reagent (supplied in TSA-direct kit; NEN Life Science Products) for 30 min, and then incubated with fluorophore or HRP-conjugated secondary antibodies diluted in TNB for 1 h at room temperature. After extensive rinsing with TNT buffer (0.1 M Tris, 0.15 M NaCl, 0.05% Tween-20), the cells were incubated with the tyramide-FITC reagent in 1× amplification diluent for 10 min to deposit FITC onto the HRP-conjugated secondary antibodies. After TNT rinses, the cultures were coverslipped with IFF mounting medium and examined using a 40× objective on a microscope (405M; Carl Zeiss, Inc. ). Antibody cross-linking (“patching”) experiments were done by incubating live retinal cultures with a polyclonal antibody against the extracellular domain of PTPμ (494) for 40 min at 37°C. The cultures were then rinsed, fixed, and permeabilized as above. The cells were processed for immunocytochemistry as above using a monoclonal antibody against N-cadherin (NCD2) or NCAM (5e) and the appropriate secondary antibodies. The double-labeled samples were examined using a 100× Neofluar objective (1.3 numerical aperture) on a confocal microscope (LSM-410; Carl Zeiss, Inc. ). Antibodies were covalently coupled to CNBr Sepharose 4B ( Pharmacia LKB Biotechnology ) using the manufacturer's protocol, or Protein A beads using a previously described protocol . E8 retinas were homogenized with a tissue tearor (200; PRO Scientific Inc.) in lysis buffer (1% Triton, 20 mM Tris, pH 7.6, 2 mM CaCl 2 , 150 mM NaCl, 1 mM benzamidine hydrochloride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mM sodium orthovanadate, 0.1 mM ammonium molybdate, 0.2 mM phenyl arsine oxide), and incubated on ice for 45 min. Triton-insoluble material was removed by centrifugation at 14,000 rpm, and the lysate was incubated with antibody-coupled beads overnight at 4°C. The beads were washed extensively with lysis buffer, and then boiled in sample buffer and separated by 6% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and immunoblotted as described . The retroviral system used is a tetracycline-repressible promoter-based (“tet-off”) system . Using the pBPSTR1 vector generously provided by Dr. Steven Reeves (Harvard Medical School, Charlestown, MA), the following constructs were generated: antisense PTPμ, sense PTPμ, and a c → s mutant form of PTPμ. A PTPμ antisense plasmid was generated that contained PTPμ coding sequence in the opposite orientation to the promoter. The sense plasmid contained almost the entire coding sequence of PTPμ . This was done to create an in-frame fusion with the green fluorescence protein (GFP) at the COOH terminus that we call PTPμGFP. This plasmid was generated by digesting full length PTPμ in bluescript and ligating it into pEGFP-N3 from Clonetech. The mutant form of PTPμ containing the cysteine-to-serine mutation at residue 1095 was generated by PCR, and a BglII/BspE1 fragment was subcloned to replace that same fragment in the wild-type form of PTPμ in the vector PTPμ/pEGFP-N3. The resulting plasmid, c → s mutant PTPμGFP, was sequenced to confirm that the single amino acid mutation was present. The c → s mutation renders the phosphatase catalytically inactive . The pEGFP plasmids containing either wild-type or mutant PTPμ were subcloned into the tetracycline-regulatable retroviral vector, pBPSTR1. A replication-defective amphotrophic retrovirus was made by transfecting the PA317 helper cell line with the respective PTPμ-containing plasmids. Control virus was generated by transfecting PA317 helper cells with the pBPSTR1 plasmid alone. The virus was added to cells in the presence or absence of 3 μg/ml tetracycline. When tetracycline was added, expression of the viral gene was inhibited. Reduction in endogenous PTPμ expression was verified by immunoblotting lysates from infected cells with antibodies to PTPμ. The density of the bands on the immunoblot films was measured using the MetaMorph image analysis program ( Universal Imaging Corp. ). Retroviral-mediated gene transfer requires that the infected cells are still mitotic in order to incorporate and express the retroviral gene. RGC neurons begin to drop out of cell division at E2–3 at a region just dorsotemporal to the optic fissure, and the wave of maturation continues outward from this region in a spatiotemporal fashion . To infect the greatest number of cells for these experiments, retinas from viral-free E3.5–4 chicks (stage 20–23) (Spafas Inc.) were used. At this age, robust neurite outgrowth occurred on N-cadherin, laminin, and L1 substrates, but not on PTPμ. Therefore, PTPμ could not be used as a test substrate for these experiments. The dissection and plating procedure is as described above except that the retinas were cut at 250-μm intervals. Explant strips from each retina were laid in alternating fashion onto two similar substrate-coated dishes, with four explants per dish, then 28 μg of polybrene and 1 ml of virus-containing medium were added for a 6–18-h incubation at 37°C. After incubation in virus, the medium was exchanged with normal culture medium. Cultures were examined at ∼24 and 48 h after plating, and neurite outgrowth from each explant was photographed. To quantify the neurite outgrowth, the 35-mm negatives were scanned and the digitized images were analyzed using the MetaMorph image analysis program. Lengths of the five longest neurites per explant were measured perpendicular to the explant tissue. To measure the number of neurites per explant, the region of neurite outgrowth was outlined to define the region of interest, the neurites were highlighted using the threshold function, and the total number of highlighted pixels per region of interest was calculated. This method provided a means to compare density between control and test conditions on each substrate. The neurite length and density measurements were analyzed by Fisher's PLSD, Scheffe, and Student's t test (Statview 4.51; Abacus Concepts, Inc.), and similar results were obtained with each of these tests for each experiment. The data from all like experiments were combined and plotted (Cricketgraph III; Computer Associates International, Inc.). During development, the retina becomes laminated such that cells of a particular type localize to distinct regions within the adult retina. A phase contrast image of an embryonic day 8 (E8) chick retina cross-section illustrates the limits of the neural retina and incomplete lamination at this stage of development . Immunohistochemical labeling of E8 retina sections showed that the greatest level of PTPμ expression was present in the retinal ganglion cell layer and in their processes that make up the optic fiber layer . RGC neurons were verified in this region by labeling with an antibody against chick L1 that specifically recognizes the RGC axons . PTPμ was also expressed at high levels in a region directly adjacent to the pigmented epithelium, which is the outer limit of the neural retina and is thought to be populated in part by mitotic precursor cells . In addition, PTPμ was observed in a region of the retina that labeled positively with the 3G3 antibody against bipolar neurons . This immunolabeling pattern is consistent with that previously described for bipolar neurons . Since the axons of RGCs form the optic nerve and are the sole output from the retina to the brain, the high expression of PTPμ on these cells is consistent with a putative role for PTPμ in neurite outgrowth. To determine whether PTPμ was capable of promoting neurite outgrowth, it was purified from brain by immunoaffinity methods using a monoclonal antibody against the intracellular domain of the protein (SK15). The PTPμ purification strategy included stringent detergent washes of the column before elution to exclude any associated proteins. The full length PTPμ protein from brain was ∼200 kD, and two fragments of ∼105 kD (predominantly extracellular form) and 100 kD (the intracellular domain, transmembrane region, and a short stretch of the extracellular domain) were observed . These fragments have been shown to be due to normal proteolytic processing of the protein into noncovalently associated extracellular and intracellular fragments, respectively . The eluted PTPμ protein was examined by immunoblotting to confirm that the preparation was not contaminated by other CAMs that promote neurite outgrowth . The samples included purified proteins from brain: PTPμ (two different preparations), N-cadherin, NCAM, and L1. N-cadherin, NCAM, and L1 were detected in the appropriate purified protein lanes, but were absent from the PTPμ preparations. PTPμ was only detected in the PTPμ preparations. These results clearly show that the PTPμ purified using these stringent conditions was not contaminated with N-cadherin, L1, or NCAM, three CAMs that are highly expressed in brain and have been previously demonstrated to promote neurite outgrowth . Retinal ganglion cell axons migrate along the surface of neuronal or glial cells in the brain during development ; therefore, CAMs expressed on the surface of these cells can serve as a substrate for neuronal migration. Due to the complexity of the developing brain, we have used an in vitro model system to gain a better understanding of the mechanisms that regulate axonal growth. The in vitro system uses a purified protein-coated dish as a substrate and measures the ability of neuronal cells to extend neurites on that substrate. The homophilic binding activity of PTPμ and expression by RGC neurons led us to test whether RGC neurons were capable of extending neurites on a PTPμ substrate. The purified PTPμ or N-cadherin preparations from brain were individually coated as a substrate on dishes and used to culture E8 chick retinal explants . Neurite outgrowth on a PTPμ substrate was observed within 2 d of plating, and the length and density of outgrowth increased over the following 2 d. Neurites grew out from explants derived from E6, 8, and 10 chick retinas, but not from E4 (data not shown), suggesting that PTPμ-dependent neurite outgrowth may be developmentally regulated. Alternatively, since the expression of PTPμ is very low at E4 , we may be unable to detect neurite outgrowth in vitro at that stage. The most robust outgrowth occurred from E8 retinas and this age was used for the experiments described here. On a PTPμ substrate, the neurites tended to be somewhat fasciculated . In addition, the growth cones were small and spiky in nature, with multiple long filopodial processes and a small lamellipodial region . In contrast, outgrowth on N-cadherin was more robust and was observed within 1 d of culture, suggesting a faster growth rate on N-cadherin than on PTPμ. Additionally, the neurites were less fasciculated on N-cadherin , and the growth cones possessed a much larger lamellipodial area and several short filopodial processes (C). The lower density of neurite outgrowth on the PTPμ substrate in comparison with the N-cadherin substrate may indicate that only a subset of RGC neurons are able to respond to PTPμ and induce neurite outgrowth. However, PTPμ was expressed at equal levels by all neurites growing on N-cadherin or PTPμ substrates (data not shown). The distinct morphology of neurites on a PTPμ substrate in comparison with N-cadherin suggests that PTPμ may use a unique signaling mechanism to promote neurite outgrowth. To ensure that the neurite outgrowth activity in the purified PTPμ preparation was mediated by PTPμ, retinal explants were cultured on a PTPμ substrate and N-cadherin or L1 control substrates in the presence or absence of function-blocking antibodies against PTPμ, N-cadherin, or L1 . Neurite outgrowth on a PTPμ substrate was completely blocked by the addition of antibodies against the extracellular portion of PTPμ , but was unaffected by the addition of antibodies against N-cadherin (C) or L1 (D). These results demonstrate that PTPμ specifically promotes neurite outgrowth. N-cadherin antibodies caused a significant inhibition in neurite outgrowth on N-cadherin . Antibodies against the extracellular domain of PTPμ had no effect on neurite outgrowth on a N-cadherin substrate . Similarly, the L1 antibodies used for these experiments completely blocked neurite outgrowth on an L1 substrate , but antibodies against PTPμ had no effect (J). Therefore, the antibodies used for these experiments were specific. Due to the longer time course to achieve neurite outgrowth on a PTPμ substrate (2–4 d), it was a concern that the retinal explants may secrete factors over time that could deposit on the dish surface and promote neurite outgrowth. For the culture of retinal explants on purified protein substrates, the dishes were first coated with substrate protein, and then the remaining binding sites on the dish were blocked by the addition of BSA before plating. A control experiment was done in which retinal explants were cultured on dishes coated with BSA alone. In this situation, no neurites were observed to extend from any explants over the course of 4 d , suggesting that the neurite outgrowth–promoting activity was due to the purified protein used as the substrate and was not due to a soluble factor originating from the retina tissue. To determine which cell types extended neurites on PTPμ, the cultures were fixed and processed for immunocytochemistry using antibodies against specific cell types of the retina. All of the long neurites growing on PTPμ were positively labeled with an antibody against L1 , which is only expressed by the RGCs , suggesting that the neurites on PTPμ were derived from RGC neurons. In addition, other cells were observed to migrate from the explants and were identified as bipolar neurons , or Müller glia . Other cells from E8 retinas, such as photoreceptor cells, were not observed to migrate on PTPμ. The cells that grew out onto the PTPμ substrate all express PTPμ, suggesting that PTPμ may be acting homophilically to promote outgrowth. PTPμ is abundant in chick retina, and its expression is developmentally regulated . PTPμ expression is observed by E4 (the earliest time-point examined), with peak expression by E11. N-cadherin is also detected in retina at E4, and undergoes a similar increase in expression with development . The molecular weight of PTPμ is altered with time, suggesting developmental regulation of glycosylation, proteolytic cleavage, and/or shedding. Similar modifications have been observed for a related RPTP, LAR . To examine whether PTPμ associates with N-cadherin, the major cadherin in retina, immunoprecipitation experiments were done using E8 retina lysates. Similar results were obtained using E4 retinas (data not shown). Both the full-length (200 kD) and cleaved (100 kD) forms of PTPμ were immunoprecipitated by antibodies to PTPμ , but not by mouse IgG . When immunoprecipitates of PTPμ were probed on immunoblots with antipancadherin antibodies, an association with N-cadherin was detected . In the reciprocal experiment, N-cadherin was immunoprecipitated by antibodies to N-cadherin , but was not detected when mouse IgG was used for immunoprecipitation (lane 1). N-cadherin immunoprecipitates contained the 200-kD full-length form of PTPμ , and the 100-kD fragment of PTPμ that was also present in the PTPμ immunoprecipitates (lanes 2 and 3). The results from the immunoprecipitation experiments indicated that PTPμ associates with N-cadherin. Since PTPμ is expressed at high levels within RGC neurons, it is likely that an association with N-cadherin occurs within these cells. To verify that PTPμ interacts with cadherins in RGC neurons, an antibody cross-linking experiment was performed using neurites from E8 chick retinal explants growing on the control substrate laminin. In Fig. 7 , A–B and E–F, the cells were fixed and processed for immunocytochemistry using the 494 polyclonal antibody against PTPμ (A and E) and a monoclonal antibody against either N-cadherin (B) or NCAM (F). In the absence of PTPμ cross-linking, N-cadherin, NCAM, and PTPμ were present in a continuous fashion along the length of the axons and growth cones. For the cross-linking experiments, the explants were cultured for 24 h to allow significant RGC neurite outgrowth, and the live cultures were incubated with a polyclonal antibody against the extracellular domain of PTPμ (494) to permit clustering of PTPμ protein on the cell surface . The cells were then fixed and processed for immunocytochemistry with antibodies to N-cadherin or NCAM (H). The PTPμ protein was cross-linked into patches along the neurites . Importantly, N-cadherin protein also became concentrated into the PTPμ patch sites . Extensive colocalization was observed between PTPμ and N-cadherin, suggesting a stoichiometric association between these proteins in neurites. In contrast, another abundantly expressed cell adhesion molecule, NCAM, did not become concentrated into PTPμ patch sites . The results of these experiments show that PTPμ and N-cadherin associate in the RGC neurites, and provide a basis for examination of the function of that association in neurite outgrowth. Based on previous studies, we hypothesized that PTPμ maintains a protein in the N-cadherin/catenin complex in a dephosphorylated state that may be important for N-cadherin–mediated adhesion and/or neurite outgrowth. To examine the role of PTPμ in N-cadherin–mediated adhesion events, a retrovirus encoding the antisense version of the PTPμ cDNA sequence was used to downregulate PTPμ expression in dissociated retinal cells. PTPμ is normally synthesized as a full length (200 kD) precursor that can be expressed at the cell surface or cleaved into a 105 kD form (predominately extracellular) or 100 kD form . Immunoblot analysis and densitometric measurement of the gel bands that were immunoreactive with an antibody against PTPμ demonstrated that the full-length protein (200-kD band) was reduced by 99% in cells infected with PTPμ antisense virus when compared with cells infected with control virus . In addition, the 100-kD band was reduced by 77%, whereas a 95-kD immunoreactive band was unchanged . These results suggest that PTPμ antisense expression inhibits the new synthesis of PTPμ. Therefore, the full-length protein made before infection has likely been cleaved over time to form the two smaller fragments that may not turn over rapidly. The retrovirus used for these experiments is negatively regulated by tetracycline. When tetracycline was present in the medium, PTPμ antisense virus had no effect on PTPμ expression level . In contrast, no change in N-cadherin expression was detected in cells infected with PTPμ antisense virus when compared with cells infected with control virus . The blot for PTPμ expression was stripped and reprobed with antibodies to NCAM to verify equal protein loading per lane . These results demonstrate that the PTPμ antisense retrovirus was able to infect the retina cells, resulting in a significant downregulation of PTPμ expression. To determine whether PTPμ function is required for N-cadherin–mediated neurite outgrowth, E4 retinas were infected with PTPμ antisense or control retrovirus. Explants from the retinas were then cultured on N-cadherin or control substrates and neurite outgrowth was examined at 24 and 48 h after plating. E4 retinas were used for these experiments because a large percentage of the cells are still mitotic at this age, which is a requirement for retroviral-mediated gene transfer. Explants infected with PTPμ antisense virus displayed a dramatic and significant decrease in neurite outgrowth on N-cadherin in comparison with sister explants infected with control virus . A range of effects was detected, including no neurite outgrowth, some short neurites, and in a few cases a small number of long neurites were observed. After infection with antisense virus, there is an ∼50% reduction in PTPμ expression in the neurites when they are immunolabeled with antibodies to PTPμ (data not shown). Therefore, there is a substantial reduction in the PTPμ phosphatase overall, but some residual expression of PTPμ in all of the neurites in the explant population. The photograph shown is representative of the median level of neurite outgrowth. Quantitation of all of the results indicated that neurite lengths were reduced by 53% and overall neurite density was reduced by 74% in cultures expressing PTPμ antisense in comparison with cells expressing control virus. In contrast, PTPμ antisense had no effect on either length or density of neurites growing on the control substrates laminin , or L1 . Since the length and density of outgrowth on laminin and L1 in the presence of PTPμ antisense virus was similar to that observed previously , it seems unlikely that the PTPμ antisense virus altered RGC differentiation. In addition, no difference in the level of axon fasciculation was observed in cultures infected with control versus PTPμ antisense virus on any substrate examined. More importantly, the ability of neurons to extend neurites on other substrates suggests that the PTPμ antisense virus was not toxic for the neurons themselves and did not have nonspecific effects on neurite outgrowth in general. These results suggest that PTPμ is specifically involved in regulating N-cadherin–mediated neurite outgrowth. The retroviral expression system used for these experiments is repressed in the presence of tetracycline . Retinal explants plated on N-cadherin in the presence of both tetracycline and PTPμ antisense virus showed no reduction in either neurite length or density when compared with explants infected with control virus. These results demonstrate that expression of PTPμ antisense is regulated by the presence of tetracycline. In addition, the observed decrease in neurite outgrowth on N-cadherin requires the expression of antisense PTPμ. PTPμ is capable of acting as both an adhesion molecule and an enzyme; therefore, it was important to determine which of these functions was necessary for the regulation of N-cadherin–mediated neurite outgrowth. To address this issue, we generated a PTPμ mutant that contained a single amino acid change (cysteine to serine) in the catalytic domain of the phosphatase. This c → s mutant, which encoded the full-length PTPμ protein tagged at the COOH terminus with GFP, preserved the adhesive function of the extracellular segment but rendered the phosphatase catalytically inactive. E4 retinas were infected with a retrovirus encoding the c → s mutant form of PTPμ, and cultured on an N-cadherin substrate. Explants infected with the c → s mutant virus displayed a dramatic and significant decrease (∼50%) in neurite outgrowth on N-cadherin in comparison with sister explants infected with control virus . These results were similar to those obtained in cultures infected with PTPμ antisense virus . Overexpression of full-length PTPμ coupled to GFP did not alter neurite outgrowth on N-cadherin . These results indicate that PTPμ tyrosine phosphatase activity is a key regulatory component for proper N-cadherin function. To gain an understanding of the function of PTPμ in the developing nervous system, we examined the ability of embryonic chick retinal neurons to extend neurites on a PTPμ substrate. PTPμ promoted neurite outgrowth from RGC neurons, and the migration of bipolar neurons and Müller glia from E8 retinal explants. Neurite outgrowth on a PTPμ substrate was blocked by the addition of antibodies against PTPμ, indicating that the neurite outgrowth activity in the purified PTPμ preparation was specific to the PTPμ protein. The morphology of neurites growing on PTPμ was unique from that observed on other purified CAMs, suggesting PTPμ may use a distinct signaling mechanism to promote neurite outgrowth. Within the retina, the expression of PTPμ is developmentally regulated and increases over time in a pattern similar to N-cadherin expression . Previous studies have demonstrated that cadherins and PTPμ associate with one another . In this study, we demonstrated that PTPμ and N-cadherin form a complex in retinal tissue. An association between PTPμ and N-cadherin was also demonstrated in RGC neurites, suggesting that PTPμ may be involved in the regulation of N-cadherin– mediated neurite outgrowth. The downregulation of PTPμ expression through antisense techniques resulted in a decreased ability of RGC neurites to extend on a N-cadherin substrate, but did not affect neurite outgrowth on laminin or L1 substrates. Overexpression of a catalytically inactive form of PTPμ also inhibited N-cadherin–mediated neurite outgrowth, thus providing further evidence that a component of the N-cadherin/catenin complex may be a substrate of PTPμ. Together, these results provide evidence that PTPμ is capable of promoting neurite outgrowth individually, and specifically regulating neurite outgrowth mediated by N-cadherin. Axonal pathfinding, fasciculation, target recognition, and synapse formation are all processes that require contact-mediated recognition of cell surface cues. The diversity of the CAMs and other molecules involved in axonal pathfinding reflects the staggering array of decisions an individual axon must make along the way to its target. Many of the guidance molecules are members of the immunoglobulin superfamily. These include CAMs like L1 and NCAM, tyrosine kinases such as Eph family members and FGF receptors and even some RPTPs; for example, DLAR, DPTP69D, and now PTPμ. Presumably, these molecules mediate specific recognition events at different points during axonal outgrowth and pathfinding. CAMs are not solely involved in adhesion of neurons to one another; they also participate in signal transduction. The interaction of a growth cone with a particular CAM can lead to rapid and specific changes in growth cone morphology . This implies the adhesion molecules are sending signals that result in a transient change in the underlying cytoskeleton that guide a neuron toward its target . Previous inhibitor studies suggested that tyrosine phosphatases are involved in the control of neurite outgrowth in general and on CAM substrates . Recent studies suggest that regulation of tyrosine phosphorylation by RPTPs affects axonal growth possibly by “steering” growth cones along the appropriate pathway . In Drosophila , two CAM-like RPTPs are expressed in the central nervous system, and knockout experiments have demonstrated that they play critical roles in development. Mutant embryos for the Drosophila RPTPs, DLAR and DPTP69D, display an inability of specific motorneurons to recognize guidance cues that allow them to innervate appropriate target muscles . In addition, a LAR homologue in leech was shown to accumulate in a subset of axonal growth cones and play a guidance role during outgrowth of these axonal processes . Together with the present study, these data provide the first evidence that RPTPs could be directly involved in axonal pathfinding and suggest that tyrosine phosphorylation is a key regulator of axonal guidance and choice point recognition. In the present study, we demonstrated that PTPμ plays a role in neurite outgrowth at physiological levels of protein expression. This data is important because previous studies on the ability of PTPμ to mediate aggregation were performed when PTPμ was massively overexpressed . We have now demonstrated that PTPμ promotes neurite outgrowth from RGC neurons, presumably through a homophilic binding mechanism. PTPμ may have several important roles in nervous system development. First, it may act as a cell–cell adhesion molecule necessary for maintenance of nervous system integrity. This could occur through homophilic binding of PTPμ on the surfaces of two apposing axons to promote axon fasciculation, a process required for nerve formation. A similar role has been suggested for other CAMs such as L1 and NCAM . Second, PTPμ may act as a permissive molecule for axonal growth. The expression of PTPμ by axons, astrocytes (our unpublished data) and other nonneuronal cells makes this role a likely possibility. A contact attraction role for PTPμ is a third possibility, such that PTPμ actively guides axons during pathfinding. For example, PTPμ may be expressed at specific choice points where axons must choose the appropriate pathway. RGC growth cones encounter several choice points during outgrowth to their target, the optic tectum . The mechanisms regulating this stereotypical innervation pattern are only partly understood, but involve tyrosine phosphorylation . A fourth possible function of PTPμ is as a sensor molecule. For example, changes in the adhesive state of the extracellular environment may be transmitted through PTPμ via regulation of its catalytic domain. PTPμ may then directly regulate the phosphorylation state of a number of cytosolic proteins, including components of the cadherin/catenin complex. This idea is supported by the inhibition of N-cadherin–mediated neurite outgrowth when RGC neurons overexpress a catalytically inactive form of PTPμ. A likely target of the PTPμ enzyme is a component of the N-cadherin complex, which was previously postulated to be a substrate of PTPμ . The association of cadherins and catenins with receptor and nontransmembrane PTPs has now been observed by many groups . The juxtamembrane domains of PTPκ and PCP2 interact with β catenin. A LAR-like RPTP associates with the cadherin/catenin complex in PC12 cells and the intracellular domain of LAR was shown to bind directly to β catenin and plakoglobin in vitro . The PTP1B cytoplasmic phosphatase was shown to interact with the N-cadherin/catenin complex and dephosphorylate β catenin , a process required for N-cadherin–mediated adhesion and actin linkage . Therefore, it is likely that regulation of the cadherin/catenin complex by PTPs will be an important mechanism of control in many cell types, including neurons. Tyrosine phosphorylation of the cadherin/catenin complex correlates with suppression of cadherin-mediated adhesion , adherens junction disassembly/ loss of cytoskeletal association , invasion and malignant progression . Tyrosine phosphorylation of the cadherin/catenin complex can be catalyzed by pp60src, EGF receptor, c-erbB2, or hepatocyte growth factor receptor . These data indicate that one of the mechanisms the cell uses to regulate the function of the cadherin/catenin complex is tyrosine phosphorylation. In the present study, PTPμ associated with N-cadherin in lysates from retina as demonstrated by immunoprecipitation techniques. A similar association was demonstrated in RGC neurites through antibody cross-linking and immunocytochemistry techniques. In addition, the ability of neurites to migrate on N-cadherin was significantly impaired when PTPμ expression was downregulated. These results provide evidence that N-cadherin–mediated neurite outgrowth requires functional PTPμ. The inhibition of N-cadherin–mediated neurite outgrowth due to overexpression of the catalytically inactive form of PTPμ further supports the idea that cadherins or their associated proteins need to be dephosphorylated to function in adhesion . Therefore, PTPμ tyrosine phosphatase activity is a key regulatory component of the N-cadherin/catenin complex. In contrast, PTPμ downregulation did not alter neurite outgrowth on L1 or laminin control substrates. These results suggest that the effects of PTPμ downregulation were specific to N-cadherin–mediated neurite outgrowth and not due to general alterations in cellular phosphotyrosine that could nonspecifically affect neurite outgrowth. Our data demonstrate that PTPμ can promote neurite outgrowth, which is consistent with a role for PTPμ in neuronal pathfinding. This promotion of neurite outgrowth could be mimicking the ability of certain neurons to respond to signaling events initiated by PTPμ. The inability of PTPμ to promote neurite outgrowth from retinas earlier than E6 suggests that a threshold level of PTPμ expression on axons may be required for PTPμ to independently promote neurite outgrowth through a homophilic mechanism. At earlier ages, PTPμ may play other specific roles; for example, regulation of N-cadherin–dependent adhesion that is required for morphogenetic movements or axonal pathfinding events. N-cadherin is one of the key molecules involved in many aspects of retinal function from histogenesis and lamination to neurite outgrowth and synapse formation . PTPμ may regulate N-cadherin function by modulating signals that allow neurons to respond to N-cadherin–mediated adhesion. | Study | biomedical | en | 0.999997 |
10087274 | Primary mammary epithelial cells were prepared from 14.5–18.5-d pregnant ICR mice as previously described . After culture for 3 days, cells were trypsinized, passed through a 70-μm nylon mesh (Falcon) to remove large cell aggregates, and plated at 1 × 10 5 cells/cm 2 in DMEM/F12 medium containing 5% fetal calf serum (Advanced Protein Products) and 5 ng/ml epidermal growth factor (Harlan Sera-Lab) overnight, then washed and cultured in serum-free DMEM/F12 medium containing appropriate combinations of 2.8 nM hydrocortisone, 880 nM insulin, 150 nM prolactin ( Sigma ), with or without inhibitors of the insulin signaling pathway. Some experiments included IGF-I or IGF-II (R&D systems) in place of insulin. The ECM substrata used for these studies were rat tail collagen I and reconstituted BM matrix prepared from Engelbreth-Holm-Swarm (EHS) tumor . Collagen I was coated onto tissue culture plates overnight at 80 μg/ml and washed extensively before use, or in some experiments commercially precoated Biocoat dishes were used ( Becton Dickinson ). A factor-reduced preparation of the BM matrix was prepared using sequential ammonium sulfate precipitations and coated onto tissue culture plastic at 14 mg/ml. In some experiments, factor-reduced BM matrix was diluted into the culture medium (final concentration 100 or 400 μg/ml) and overlaid onto first passage cells plated on collagen I . To analyze DNA integrity, first passage cells were cultured in medium containing appropriate hormones and inhibitors for two days, then washed over a period of 2 h to remove the accumulated dead cells and debris. Fresh medium was added to cells and after 4 h newly apoptotic cells were collected from the medium and pooled with any remaining attached cells. DNA was extracted from samples , its OD was measured at 260 nm, and separated on a 1% agarose gel to confirm DNA loading after staining the gel with ethidium bromide. Equal amounts of DNA were separated by conventional agarose gel electrophoresis, southern blotted, and apoptotic DNA ladders were visualized by hybridization with a digoxigenin-labeled total mouse genomic DNA probe ( Boehringer Mannheim ). Single cell cultures were prepared by straining first passage cells through a 20-μm mesh, pelleting and resuspending in factor-reduced BM matrix at 2–4 × 10 6 cells/ml, as described . 100 μl of the mammary cell suspension was gelled for 60 min on dishes precoated with a thin layer of BM, then cultured in serum-free DMEM/F12 medium containing appropriate hormones, inhibitors or antibodies for 2 d before fixation in 2% paraformaldehyde. 25-μm cryosections were stained with 0.5 μg/ml Hoechst 33258 and apoptotic nuclei were counted in the single cell population. Each experiment was repeated two to three times and in each experimental condition, >600 single cells were scored for apoptosis. Polyclonal rabbit anti–β1 integrin antibody was prepared in this laboratory . It was shown to inhibit the adhesion of first passage mouse mammary epithelial cells to laminin, collagen IV, but not fibronectin or vitronectin. Purified rat monoclonal antibody to α6 integrin (GoH3) and its rat IgG2a isotype control were obtained from Serotec. Rabbit polyclonal anti-laminin antibody was raised against purified laminin isolated from EHS tumor (a kind gift of P. Yurchenco, Robert Wood Johnson Medical School, Piscataway, NJ). This laminin had previously been shown to contain laminin A and B chains but not collagen IV or nidogen . A protein A–derived IgG fraction from the rabbit serum was subsequently purified on an EHS affinity column. This antibody significantly inhibited the adhesion of first passage mouse mammary epithelial cells to laminin at 5 μg/ml and completely inhibited adhesion at 30 μg/ml (data not shown). A quantitative assay for apoptosis based on measuring cellular detachment from different substrata was previously described . Cells detaching into the medium of first passage cultures over periods of 4 h were pelleted and counted using either a hemocytometer or a Coulter counter. Detached cells were confirmed to have apoptotic morphology by fluorescence microscopy after staining with 0.5 μg/ml Hoechst 33258, and the remaining monolayers were also stained with Hoechst 33258 to confirm that apoptotic cells did not accumulate there. Quantitative data for detachment assays are all relative to the number of cells that attached to each substrata after plating. Mammary cells were cultured for 2 d, then washed and serum-free DMEM/F12 medium with or without insulin was replaced for 15 min. The cells were washed twice with 1 mM Na 3 VO 4 , 10 mM NaF in PBS and scraped into NET lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 10 mM NaF, 1 mM Na 3 VO 4 , 10 μg/ml aprotinin, 20 μM leupeptin, 1 mM PMSF, pH 7.5). Cells were lysed by vortexing and then rotating for 30–60 min at 4°C, and detergent insoluble proteins were cleared by centrifugation at 7,000 g . Aliquots of samples were subjected to SDS-PAGE on 6% gels, and the gels were stained with Coomassie Brilliant Blue to normalize protein levels. Equal amounts of protein were immunoprecipitated either with anti–IRS-1 antibody (UBI) or anti-insulin receptor β antibody (Transduction Laboratories) and 20 μl protein A–Sepharose beads ( Zymed Laboratories ), before being separated on 6% gels under reducing conditions and transferred to Immobilon-P membranes ( Millipore ). Phosphotyrosine blots were blocked in 1% BSA in TBST, (50 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) and incubated overnight with monoclonal antibody 4G10 (UBI) at 1:1,000 for insulin receptor β or 1:3,000 for IRS-1. Interaction of the p85 subunit of PI 3-kinase with IRS-1 was examined by probing IRS-1 immunoblots with anti–PI 3-kinase (UBI). For each experiment, equal amounts of protein were confirmed for IRS-1 by blocking IRS-1 immunoblots in 3% nonfat dry milk/PBS and probing with 1:3,000 anti–IRS-1 antibody; for insulin receptor β, whole cell lysate blots were blocked with 3% BSA/TBST and probed with 1:250 anti-insulin receptor β. Protein kinase B (PKB, otherwise known as c-Akt) was analyzed by immunoblotting equal amounts of cell protein with either an anti-PKB antibody or an antibody specific for phosphorylated PKB (both at 1:2,000; New England Biolabs ). Proteins were visualized by enhanced chemiluminescence ( Amersham ). Mammary epithelial cells require appropriate cell–ECM interactions for survival since they undergo apoptosis in the absence of a BM . To determine the mechanism by which BM suppresses apoptosis, we asked whether BM was able to act as a survival ligand by itself or whether additional factors were required to prevent cell death. First passage mammary epithelial cells isolated from midpregnant mice were plated on substrata of collagen I or a reconstituted BM matrix derived from the EHS tumor. Culture on a BM matrix in the presence of lactogenic hormones, insulin, prolactin, and hydrocortisone, suppressed apoptosis, whereas cells died on collagen I . However, removal of lactogenic hormones from the medium resulted in apoptotic death of the cells, even those cultured on the BM matrix . This indicated that survival of mammary cells required two signals, one from the BM and a second signal from the hormonal milieu. To determine which of the three hormones acted in a survival capacity, cells plated on BM were incubated with combinations of insulin, prolactin, and hydrocortisone and the extent of cell death was measured by DNA fragmentation. Apoptosis occurred only when the cells were cultured without insulin . We also used an alternate assay for apoptosis, where single mammary cells were cultured within a BM gel, but apart from each other so that they were unable to form cell–cell interactions . In this assay, apoptosis was measured by determining nuclear morphology after culture for 48 h . 20–30% of single cells were apoptotic providing insulin was present in the medium. However, in the absence of insulin, cell death increased significantly ( P < 0.01) to 60– 70% . These two different approaches collectively demonstrate that BM does not act alone to suppress apoptosis in primary cultures of mammary epithelia. Instead, it regulates survival in combination with signals elicited by insulin. Furthermore, the data show survival is not dependent on mammary differentiation, since removal of prolactin and hydrocortisone, which are required for milk protein gene expression, did not result in apoptosis. In addition to the ability of insulin to interact with its own receptor, this hormone can also bind to the IGF receptor although with a 100–1,000-fold lower affinity . IGF-I or IGF-II have been shown to act as survival factors in other cell types , and we therefore tested whether they could directly suppress mammary apoptosis. At physiological concentrations, both IGF-I and IGF-II significantly inhibited the DNA fragmentation exhibited by cells cultured on a BM matrix in the absence of other hormones . Moreover, IGF-I suppressed apoptosis ( P < 0.01) in single mammary cells cultured within the BM gel to the same extent as insulin . The survival of mammary epithelia can therefore be regulated by signals from IGF-I and IGF-II in addition to those from insulin, and these signals act coordinately with BM. Mammary epithelial cells plated onto a BM substratum form multicellular, alveolar-like structures . Thus, it was important to ask whether this complex three-dimensional structure was involved with mammary cell survival, or if signals from the BM–integrin interactions were sufficient. Therefore, we cultured mammary cells as monolayers on collagen I and incubated them with the EHS BM preparation diluted into the culture medium. Under these conditions, the cells did not form alveoli but remained as monolayers on the culture dish with some matrix proteins precipitating over the cells . Dilution of the BM preparation 140-fold to 0.1 mg/ml resulted in significant suppression of apoptosis, both in a quantitative assay and in DNA fragmentation studies . BM proteins diluted 35-fold to 0.4 mg/ml suppressed apoptosis to virtually the same extent as in the cells cultured as alveoli on top of a BM substratum . Thus, the three-dimensional multicellular structure is not a primary determinant of mammary cell survival. BM contains several ECM proteins and one component, laminin, has previously been shown to be a survival ligand for other cell types . To determine whether laminin was a survival factor for mammary epithelial cells we developed a function-blocking anti-laminin antibody that inhibited adhesion of mammary cells to laminin (data not shown). We assessed whether this antibody was able to interfere with the survival of mammary cells that had been cultured as single cells within BM gels . We found that when the anti-laminin antibody was included in the gel, mammary cells underwent apoptosis even when they were cultured in the presence of insulin. In contrast, an adhesion-blocking antibody directed against fibronectin had no effect , neither did the inclusion of an RGD peptide in the assay . These results demonstrated that within the context of a three-dimensional BM gel, laminin has a survival-promoting function. Indeed, they show that both insulin and laminin can act as survival ligands for mammary epithelial cells, although they do not rule out the possibility that other soluble factors or ECM proteins may have a similar function. Major cellular receptors for BM proteins, including laminin, are the transmembrane heterodimeric integrin receptors. To confirm that BM acts directly to suppress apoptosis, and that the survival signals were mediated by integrins, we assessed whether function-blocking anti-integrin antibodies interfered with the mammary cell survival using the single cell assay. In experiments where an adhesion-blocking polyclonal anti–mouse β1 integrin antibody was included in the gel, insulin failed to suppress apoptosis. Indeed, the levels of death were similar to those observed in single mammary cells in the absence of insulin . One receptor for laminin is α6β1 integrin, and we therefore performed parallel experiments using a function-blocking anti–α6 integrin antibody . The cellular response was similar to that for the anti–β1 integrin antibody, providing evidence that both the α6 and β1 integrin subunits can transduce survival signals in normal mammary epithelial cells. Suitable antibodies to other mouse integrin subunits were not available to test for specificity. Therefore, we performed similar experiments using function-blocking anti–human integrin antibodies and demonstrated that α6 and β1 integrin, but not α2 integrin, acts as survival receptors in primary cultures of human mammary cells (Oliver, J., M. O'Hare, and C.H. Streuli, manuscript in preparation). In addition, our results with anti-fibronectin antibodies and RGD peptides do not support a role for αv integrins in mammary cell survival. Our combined data using function-blocking antibodies to laminin and to integrin subunits show that suppression of apoptosis in mammary epithelia depends on interactions of the cells with BM through the α6 and/or β1 integrin receptors, and that laminin can act as a survival ligand. Since both insulin and laminin had been identified as mammary cell survival factors, it is possible that they trigger separate pathways required for suppressing apoptosis. However, recent work on the control of cell cycle and differentiation indicates that ECM affects the ability of growth factors/cytokines to trigger their downstream signaling kinases . Thus an alternative mechanism to explain survival signaling in mammary cells is that the insulin- and BM-triggered pathways converge. To determine whether this was indeed the case, the extent of tyrosine phosphorylation in the proximal components of the insulin signaling pathway was measured . Insulin interacts with its receptor, causing receptor oligomerization and activation of the kinase domain. This results in tyrosine phosphorylation of the receptor β subunit. The adaptor protein insulin receptor substrate-1 (IRS-1) is recruited to the insulin receptor, then becomes tyrosine phosphorylated creating SH2 domain interaction sites for downstream enzymes such as PI 3-kinase . The ability of insulin to signal in primary cultures of mammary cells plated on BM was compared with those on collagen I. Tyrosine phosphorylation of the insulin receptor β subunit occurred within 15 min of insulin treatment and was independent of cell– ECM interactions . However, the ability of IRS-1 to become tyrosine phosphorylated in response to insulin was strongly dependent on cell interactions with BM, and virtually no IRS-1 phosphorylation was observed in cells cultured on collagen I . It has been shown in several cell types that PI 3-kinase is required for preventing apoptosis, and that in neuronal cells and fibroblasts IGF-I suppresses apoptosis through PI 3-kinase . PI 3-kinase is activated in response to insulin or IGF-I following the interaction of its p85 subunit with IRS-1, and therefore we assessed whether cell–ECM interactions regulated the association of the p85 subunit of PI 3-kinase with IRS-1 . PI 3-kinase did not associate with IRS-1 in mammary cells cultured on either substratum in the absence of insulin . However, within 15 min of insulin treatment PI 3-kinase bound IRS-1, but the extent of the interaction was strongly dependent on cell adhesion to BM; quantitation of the data indicated that threefold more PI 3-kinase bound to IRS-1 in cells cultured on a BM substratum than in cells cultured on collagen I . One downstream target of PI 3-kinase signaling pathway is PKB, which has previously been implicated in the suppression of apoptosis . This enzyme is recruited to the plasma membrane following PI 3-kinase phosphorylation of membrane lipids, and then activated phosphorylation on serine/threonine residues . Using an antibody specific for phosphorylated PKB, we found that maximal activation only occurred when mammary cells treated with insulin had been cultured on BM . Wortmannin completely abrogated insulin-induced PKB phosphorylation. Since many of the assays to measure mammary apoptosis in our work were carried out over several days, we also measured PKB phosphorylation status in cells cultured with insulin for 3 days. PKB was only phosphorylated in cells cultured on BM, even though its level remained constant whether the cells were on collagen I or BM substrata. These results are an indirect confirmation that PI 3-kinase activity depends on coordinate signals from insulin and BM. Most importantly, they demonstrate that the PKB pathway, already shown to be a critical determinant of survival in other cell systems, relies on converging insulin and ECM signals in mammary epithelia. Thus, the ECM dependence of insulin signaling correlates with the ability of BM, but not collagen I, to deliver survival cues. The experiments described above suggested that the mechanism through which cell–ECM interactions regulate apoptotic fate in mammary epithelial cells is through a control on insulin-mediated PI 3-kinase signaling. To determine whether the PI 3-kinase pathway, or related pathways, were required for survival, the effects of the pharmacological inhibitors LY 294002 and wortmannin were examined in two independent assays for mammary apoptosis. Mammary cells were cultured on a BM matrix with 10– 100 nM IGF-I, with or without 0.1 or 1 μM LY 294002. In the absence of soluble factors the cells underwent apoptosis , and this was suppressed by IGF-I . However, IGF-I failed to suppress apoptosis when LY 294002 was included in the cultures . We observed low levels of apoptosis at 0.1 μM and very extensive DNA fragmentation at 1 μM LY 294002, a concentration previously shown to inhibit IGF-I signaling and PI 3-kinase activity . Similar results were obtained with wortmannin (data not shown). The effect of the kinase inhibitors on suppression of apoptosis by insulin was also examined in the single cell assay. As before, mammary cells cultured within BM matrix were not able to survive in the absence of insulin. Insulin rescued the cells from apoptosis, but low levels of wortmannin (1 μM) and LY 294002 (0.1 μM) blocked survival signaling . The target specificity for wortmannin is fairly broad but includes PI 3-kinase whereas LY 294002 is specific for PI 3-kinase . Thus, these two experiments indicate that the PI 3-kinase class of enzymes are necessary for mammary cell survival. Together our results suggest that mammary epithelial cell survival depends upon a functional PI 3-kinase signaling pathway which is normally triggered by insulin when the cells are cultured with a BM substratum. Restricting the activity of this pathway by culture on collagen I results in cell death. The mechanisms that regulate death and survival decisions in epithelial cells are not understood. Yet these are fundamental cellular processes and are essential not only for deciphering the control of normal development, but also for designing strategies to tackle progressive diseases such as breast cancer. In this study, we have addressed the mechanism whereby BM suppresses apoptosis in primary cultures of mammary epithelial cells and found that this type of ECM controls the ability of insulin to deliver survival signals. We have demonstrated that insulin is necessary to prevent death of mammary cells through the PI 3-kinase pathway, and furthermore that insulin-mediated interaction of PI 3-kinase with IRS-1 and phosphorylation of PKB, a key enzyme downstream in the PI 3-kinase pathway, is influenced by cell interactions with BM. This provides new evidence for the importance of ECM in the growth factor–mediated regulation of cell phenotype. Since α6 and β1 integrins are necessary for mammary cell survival, our work supports the notion that one function of integrins is to regulate growth factor signaling. Our previous studies, and those of others, have demonstrated that mammary epithelial cells depend on BM for survival . We have now found that they require a further signal from soluble ligands to remain alive. Of three hormones tested, insulin, but not hydrocortisone or prolactin, was found to be necessary for suppression of apoptosis . This survival effect of insulin could be mimicked by IGFs. Insulin and IGFs have been shown to rescue apoptosis in several cell lines following interleukin-3 or serum withdrawal . However, although IGFs have been proposed to act as survival factors in mammary gland, this has not been demonstrated directly. For example, overexpression of des(1-3)hIGF-I or IGF-I in mammary glands of transgenic mice delayed involution and the glands showed lower levels of apoptosis, but the target cells were not identified . In the experiments performed in our study, purified populations of mouse mammary epithelial cells survived in the presence of insulin or IGFs, indicating that the factors suppress apoptosis by a direct action on the epithelial cells themselves. Glucocorticoids have also been demonstrated to rescue cells from apoptosis in the mammary gland, although it was not determined whether the hormone acts directly on epithelial cells or through an indirect mechanism . In purified mammary epithelial cells hydrocortisone had a minimal effect on survival, and therefore its function in vivo would appear indirect. Thus, although in vivo studies have indicated a role for both IGFs and glucocorticoids in suppressing apoptosis in mammary gland, our study with isolated cells shows that only IGFs act directly on the epithelial cells themselves. IGFs are normally only synthesized by stromal cells in the mammary gland, but their expression is regulated by a synergy between growth hormone and estradiol . Therefore, one explanation for the inhibition of apoptosis by hydrocortisone in vivo is that this steroid can act on stromal cells to alter IGF expression. Alternatively, since IGFBP-5 has been suggested to induce mammary involution, glucocorticoids may inhibit apoptosis in vivo by decreasing IGFBP-5 synthesis, as occurs in cultured fibroblasts . The ability of prolactin to suppress apoptosis directly in purified mammary epithelial cells was also tested. In agreement with previous studies which showed that prolactin could not rescue mammary apoptosis in vivo, we demonstrated that it also had a negligible effect on epithelial cell survival in culture . Prolactin regulates intracellular signaling through a Jak/Stat pathway, which is required for mammary differentiation. Although other cytokines have been proposed to suppress apoptosis through Stat transcription factors, it appears that this pathway is not directly involved in mammary cell survival . Together our data show that, of the hormones tested, insulin is sufficient for the survival of mammary cells cultured on BM. Since prolactin and hydrocortisone, in addition to insulin, are necessary for lactation, the results indicate that mammary cell survival does not depend on differentiation. Moreover, as cells on collagen I undergo apoptosis even in the presence of all three hormones, the survival mechanism would appear to have more to do with cell–ECM interactions than with differentiation. Cell–ECM interactions are known to suppress apoptosis in epithelial cells . However, such studies are based on comparing the survival potential of suspension-cultured epithelial cell lines with those adhering to ECM in short term assays. We have developed a model where primary mammary cells isolated directly from tissue are maintained on physiological substrata in longer term assays. These cells have not been selected for their ability to form cell lines and therefore retain an apoptotic potential that is as close as possible to that in vivo. They undergo apoptosis specifically after culture for several days on tissue culture plastic or collagen I, but not on BM . The cells plated on a BM substratum form multicellular structures resembling alveoli in vivo . However, this three-dimensional architecture did not appear to play a role in suppression of apoptosis . Under conditions where the cells remained as monolayers, apoptosis was efficiently suppressed by diluted BM proteins precipitating over the cells. Further verification that BM had a direct survival signaling role came from studies using function-blocking antibodies. Using a single cell assay developed previously for analysis of differentiation , we found that although insulin was able to significantly suppress apoptosis in cells cultured within a three-dimensional BM gel, it was not able to do so if anti-laminin antibodies were included . Laminin has previously been shown to act as a survival ligand for other cell lineages including fibrosarcoma and neuroblastoma cells and has been implicated in preventing apoptosis during myogenesis . Our results extend these previous findings and show that laminin also has a survival function in mammary epithelium. However, laminin may not be a survival ligand for all cells, since endothelial cells underwent apoptosis on laminin substrata but not on fibronectin or vitronectin . Thus, distinct cell types have a different requirement for ECM to rescue them from apoptosis. It remains to be determined whether or not the survival response to different ECM ligands is mediated through similar signaling pathways. In several cell systems, integrins have been shown to be required for mediating a survival response . We tested the possibility that integrins delivered survival signals in mammary cells by examining the survival of single cells in BM gels after treatment with function-blocking antibodies, and found that both α6 and β1 integrins were necessary to prevent the cells from undergoing apoptosis. Although β1 integrins have been shown to be required for mammary epithelial cell survival, our results augment these studies by providing the first direct evidence that α6 integrin functions in such a pathway . These findings have recently been confirmed in our experiments with primary human breast epithelia, where anti–α6 and anti–β1 integrin antibodies induced cell death, although antibodies to the α2 subunit did not. The latter result indicates a specificity of response as mammary cells express significant levels of α2 integrin, but it also suggests that the α2β1 integrin, a receptor for collagen, is not involved with suppressing mammary apoptosis. The β1 and β3 integrin subunits have previously been shown to be survival receptors . RGD is a peptide substrate for αvβ3 integrin but we found that it had no effects on mammary cell survival. Furthermore, in preliminary immunostaining experiments with anti–αv integrin antibodies, we have only detected this subunit in the epithelial cells of virgin mammary gland and it appeared to be completely absent from pregnant and lactating tissue. Therefore, our data would not support the possibility that it is a survival receptor for mammary cells isolated from pregnant mammary gland, even though it does have this role in endothelia . In addition to the β1 integrin subunit, mammary cells also express β4 integrin, and both of these can partner the α6 subunit. Therefore, our data are not yet sufficient to confirm that laminin suppresses apoptosis through a direct cell interaction with the α6β1 integrin heterodimer, neither do they exclude the possibility that other integrin subunits are involved in survival. However, they do demonstrate that laminin is a ligand for survival and that the α6 and β1 subunits can act as survival receptors. The metastatic breast cancer cell line, MDA-MB-435 expresses α6β1 integrin. Tumors induced by a derivative cell line in which α6β1 was functionally ablated were much smaller those of the parental cells, and showed a sixfold higher apoptotic/mitotic index . These results suggest that α6 integrin provides an anti-apoptotic signal in vivo as well as in culture and together with the present study, they may explain why higher levels of α6 integrin in human breast carcinomas correlate with an increased likelihood of patient morbidity . Moreover, they suggest that α6 integrin–ligand interactions might represent a target for therapeutic intervention in breast disease, especially in combination with agents which inhibit growth factor–mediated survival signals. Since both BM and insulin were required for sustained prevention of apoptosis in mammary cells, it was possible that these ligands triggered either parallel or convergent survival pathways. An abundance of studies indicate that PI 3-kinase is essential for suppressing apoptosis in other cell systems, possibly through PKB which has been implicated as a downstream regulator of survival . Our experiments using inhibitors also demonstrated a requirement for PI 3-kinase in mammary cell survival . Therefore, we examined whether this pathway was independently regulated by the two separate ligands or if BM controlled the ability of insulin to trigger its phosphorylation cascade, by measuring the levels of phosphotyrosine in its proximal signaling proteins. Insulin rapidly induced tyrosine phosphorylation of its receptor. However, the signaling events downstream of the insulin receptor resulting in IRS-1 tyrosine phosphorylation and its association with PI 3-kinase were only propagated in cells cultured on BM and not on collagen I . These results contrast with a study using CHO cells overexpressing insulin receptor, which showed that transient adhesion to fibronectin >20 min enhanced the insulin-induced phosphorylation of both the receptor and IRS-1 . Our data support the conclusion that ECM amplifies insulin signaling, but extends it by showing that once nontransfected primary epithelial cells have become established within an ECM environment over several days, they develop a selective sensitivity to growth factor signaling. Thus, the cellular environment determines whether the insulin signal can be propagated efficiently, and this only occurs when the cells contact a BM but not when they are on collagen I. Indeed, the results indicate the existence of an ECM-dependent restriction point in insulin signaling, which occurs downstream of insulin receptor phosphorylation. The conclusion that BM and insulin cooperate to drive insulin signaling was confirmed in further experiments where we examined PKB phosphorylation, using both short and long term treatments with insulin . Cell– matrix interactions had a similar effect of enhancing EGF signaling in MDCK cells, although in that study the PKB activation was examined in cells briefly attached to an ECM in comparison with those detached from a substratum . Together, these complementary results indicate that both the short and long term activation of enzymes in the PI 3-kinase signaling pathway requires adherent cells to be in the correct ECM microenvironment, which contributes to both EGF and insulin signaling. At the current time we do not know whether PKB is absolutely required for mammary cell survival, but given the results from other cell systems, this seems likely. Candidates for the downstream link between PKB and the apoptotic machinery include glycogen synthase kinase-3, procaspase-9, and the pro-apoptotic protein Bad . Although the phosphorylation of PKB and Bad have been dissociated from each other, primary cultures of mammary epithelial cells do express Bad, which therefore remains a potential target for regulation by PKB . Adhesion to ECM has previously been shown to suppress apoptosis in several cell types . In addition, cell–ECM interactions trigger integrin-mediated downstream phosphorylation cascades involving MAP kinase and PI 3-kinase, providing possible mechanisms for ECM control of survival . However, in the mammary gland model we have now demonstrated that although integrin signaling is necessary for survival, sustained cell interactions with a BM substratum alone are not sufficient to suppress apoptosis. In addition, insulin or IGFs are also required. Similar conclusions have been reached in studies of the cell cycle where serum factors and ECM are both required for MAP kinase activation, cyclin E–cdk2 activation, and retinoblastoma phosphorylation . Dual signals from soluble factors and ECM are also implicated in the control of mammary differentiation, where prolactin and BM are both necessary for triggering the prolactin signaling cassette, Stat5 DNA binding, and milk protein gene transcription . Although our work shows that both laminin and insulin act as survival ligands for mammary cells, we cannot rule out the possibility that other types of ligand are important in regulating survival. Insulin, for example, was only able to suppress apoptosis of single mammary cells cultured within BM by about twofold, suggesting that other factors may play a role in mammary cell survival in vivo. Preliminary studies with primary cultures of human mammary epithelial cells, have demonstrated that apoptosis in the single cell assay can be reduced by insulin, but is further suppressed by EGF. But with mouse mammary cells, EGF does not have an additional survival effect over and above insulin (Oliver, J., and C.H. Streuli, unpublished data). Therefore, it may be that additional cytokines can suppress apoptosis still further than insulin and BM in the mouse system. Even if other mammary survival factors remain to be identified, our data are still sufficient to demonstrate that both insulin and integrin contribute to the survival of mammary cells. In summary, these studies indicate that an important function of cell–ECM interactions is to modulate growth factor and cytokine responses. This suggests that current thinking about growth factor signaling in adherent cells should include a component from the ECM. One mechanism might be via direct or indirect associations between integrin and growth factor receptors, as has been demonstrated for αvβ3 integrin and the insulin and PDGF receptors, or with integrin and downstream components in the insulin signaling pathway, as shown with αvβ3 integrin and IRS-1 . The role of integrins and the cytoskeleton in signal transduction may be to provide a scaffold whereby components of growth factors cassettes are assembled into discreet domains within the cell, so that they can propagate signals efficiently. Indeed, a modular nature for membrane signaling complexes has been proposed, which may explain the recruitment of signaling proteins to cytoskeletal structures such as adhesion plaques . Our current goal is to address this issue by determining whether the macromolecular organization of insulin signaling proteins and integrins in mammary cells cultured on BM are different to those in cells on collagen I. | Study | biomedical | en | 0.999995 |
10087275 | Genomic clones containing the ColQ gene were isolated by screening a 129sv strain mouse genomic library (Stratagene) with cDNAs encoding rat ColQ . For targeting vector PRAD1 , a 0.8-kb EcoRI-HindIII fragment containing the exon encoding the PRAD domain as well as adjacent intronic sequences was replaced by a PGK- neo cassette . For targeting vector PRAD2 , a 0.6-kb HindIII fragment containing the PRAD exon was replaced by a cassette containing neo plus the Escherichia coli lacZ gene, for monitoring gene expression. The PRAD2 vector also contained a diphtheria toxin-A gene for negative selection . Both constructs were transfected into R1 ES cells by electroporation. Homologous recombinants were identified by PCR and confirmed by probing genomic Southern blots with an 0.8-kb fragment of the ColQ gene that was entirely external to the targeting vector . Chimeric mice from one PRAD1 and two PRAD2 ES cell clones gave rise to heterozygous and then homozygous mutant mice. The PRAD2 vector contained a lacZ gene, but no β-galactosidase activity was detected in heterozygotes, either histochemically or immunohistochemically. For immunohistochemistry, sternomastoid and tibialis anterior muscles were embedded in Tissue-Tek OCT compound (Sakura Finetek USA), frozen in liquid nitrogen–cooled isopentane, and sectioned at 10 μm in a cryostat. Sections were blocked for 1 h with 2% BSA and 5% normal goat serum in PBS, incubated with primary antibodies for 1–2 h, washed, and then reincubated with secondary antibodies for another 1–2 h. Antibodies to the following antigens were used: ColQ , AChE (a gift of Terrone Rosenberry, Mayo Clinic), α-sarcoglycan, β-dystroglycan, utrophin (Novocastra Laboratories Ltd.), rapsyn , and agrin (a gift of Z. Hall, University of California, San Francisco). FITC-conjugated Vicia villosa agglutinin-B4 (VVA-B4) was obtained from Sigma Chemical Co. and rhodamine-α-bungarotoxin was obtained from Molecular Probes. Cholinesterase activity was detected by the histochemical method of Karnovsky and Roots on sections fixed with 2% paraformaldehyde in PBS. To distinguish AChE from BuChE, the reaction mixture was supplemented with 10 −4 M tetraisopropylpyrophosphoramide (iso-OMPA), a selective inhibitor of BuChE, or with 5 × 10 −5 M 1:5-bis (4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51), a selective inhibitor of AChE . Where indicated, sections were treated with 1% Triton X-100 for 60 min at room temperature or highly purified bacterial collagenase (800 U/ml, type VII; Sigma Chemical Co. ) for 60 min at 37°C before fixation and staining. For electron microscopy, mice were perfused with lactated Ringer's solution followed by 4% paraformaldehyde and 4% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2. Sternomastoid muscles were dissected and fixed overnight at 4°C. The endplate-rich region of the muscle was refixed in 1% OsO 4 in cacodylate buffer, dehydrated, and embedded in Araldite. Thin sections were stained with uranyl acetate and lead citrate. Mice were killed and tissues were homogenized in a glass Potter homogenizer in 1 ml of low-salt (LS) buffer, which contained 50 mM Tris, pH 7.0, 40 mM MgCl 2 , 1% Triton X-100, 2 mM benzamidine, 40 μg/ml leupeptin, and 25 μg/ml pepstatin. The homogenates were centrifuged for 20 min at 60,000 rpm, the supernatant was removed, and the pellet was homogenized in a second aliquot of LS buffer and centrifuged again. The two resulting supernatants comprised the detergent-soluble (DS) extracts. The pellet was then homogenized with 300 μl of high-salt (HS) buffer, which consisted of LS buffer plus 0.8 M NaCl. After centrifugation, the supernatant (HS extract) and the pellet were both saved. Samples of the DS and HS extracts were analyzed by sedimentation in 5–20% sucrose gradients containing 50 mM Tris, pH 7.0, 40 mM MgCl 2 , 1% Brij96, and 0.4 M NaCl. Centrifugation was in an SW41 rotor for 16 h at 39,000 rpm. About 75 fractions of 150 μl were collected from the bottom of the tube. 60-μl aliquots were transferred to a microtiter plate. Cholinesterase activity was assayed colorimetrically, using acetylcholine as substrate . To assay AChE, iso-OMPA (10 −4 M) was included to inhibit BuChE. To assay BuChE, BW284c51 (10 −6 M) was included to inhibit AChE. Alkaline phosphatase (6.1 S) and β-galactosidase (16 S) from E . coli were included as internal sedimentation standards, and their activity profiles were used to establish a linear relationship between the fraction numbers and Svedberg units . All reagents were from Sigma Chemical Co. ColQ −/− mice or control littermates were killed and immediately exsanguinated. Hemi-diaphragms with their associated phrenic nerves were isolated. The two hemi-diaphragms were separated and each was mounted in a 2-ml bath lined with Rhodorsil (Rhône-Poulenc) and superfused with saline of the following composition: 154 mM NaCl, 5 mM KCl, 2 mM CaC1 2 , 1 mM MgCl 2 , 5 mM Hepes, pH 7.4, and 11 mM glucose. The solution was bubbled with pure O 2 . The membrane potential and miniature endplate potentials (mepps) were recorded from endplate regions at room temperature (22–24°C) with intracellular microelectrodes filled with 3 M KCl (resistance of 16–26 MΩ), using conventional techniques and an Axoclamp-2A system (Axon Instruments). Recordings were made continuously from the same endplate before and throughout application of the cholinesterase inhibitors tested. Data were digitized and analyzed using a computer equipped with an analogue and digital I/O interface board and a program kindly provided by Dr. John Dempster (University of Strathclyde, Scotland). Mepps were analyzed individually for amplitude and time course. Clones encoding the ColQ gene were isolated from a genomic library and used to produce two targeting vectors, PRAD1 and PRAD2 . Both vectors deleted the PRAD domain of ColQ, which forms the binding site for the catalytic (AChE T ) subunits of AChE . Both targeting vectors gave rise to homologous recombinant ES cells, germline chimeras, heterozygotes, and homozygotes . Phenotypes of PRAD1 and PRAD2 homozygotes were identical, so both are called ColQ −/− and described together here. Homozygous ColQ −/− mice were born in expected numbers, and were externally indistinguishable from heterozygous ( ColQ +/− ) and wild-type siblings at birth. Around 5 d after birth (postnatal day [P]5), however, ColQ −/− mice began to exhibit a tremor when moving. The tremor persisted throughout life. The homozygotes remained active, responsive, and capable of feeding, but failed to thrive, and grew at a slower rate than littermates . About half of the ColQ −/− mice died at about the time of weaning (P21) and around two-thirds of the survivors died over the next few weeks. 10–20% of ColQ −/− mice lived into adulthood (>3 mo) but remained much smaller than littermates. Sections of skeletal muscle from ColQ −/− mutants and littermates were doubly stained with an antiserum to recombinant ColQ plus rhodamine-α-bungarotoxin, which binds to AChRs and thereby marks synaptic sites. In controls, ColQ was concentrated at synaptic sites and was undetectable extrasynaptically . In ColQ −/− mutants, AChR-rich synaptic sites were nearly normal in size, shape, and distribution (see below) but, as expected, bore no ColQ . The ColQ subunit is believed to anchor AChE to the basal lamina at the neuromuscular junction. To obtain definitive evidence on this point, we stained sections of mutant and control muscle with antibodies specific for the AChE catalytic subunit. This antibody recognizes both of the known alternatively spliced products (T and H) of the mammalian AChE gene . AChE was abundant at all synaptic sites in controls, but undetectable in homozygotes . This result suggested that ColQ is required for accumulation of all synaptic AChE. An alternative explanation, however, was that the association of AChE with synaptic membranes or matrix had merely been weakened in the absence of its collagenous tail, in which case AChE might have been present at synapses in vivo but lost during preparation of sections. Therefore, we performed two additional tests to seek AChE at synaptic sites in ColQ −/− muscle in situ. First, we injected fasciculin-2, a selective inhibitor of AChE into ColQ −/− mice and their littermates. This inhibitor was chosen because it penetrates the central nervous system poorly, and is therefore believed to exert its effects predominantly on peripheral AChE. Fasciculin-2 (3–6 μg/g, injected intraperitoneally) caused tremor followed by flaccid paralysis in controls. In mutants, in contrast, effects of fasciculin were minor, and injected animals were reflexive and capable of righting. Second, we recorded mepps from muscle fibers of ColQ −/− mice and littermate controls . The decay times of mepps were variable but on average slower in homozygotes than in controls (data not shown), presumably because AChE terminates neurotransmitter action at normal neuromuscular junctions , whereas diffusion does so in ColQ −/− muscle. More importantly, addition of fasciculin greatly slowed the decay of mepps in normal muscle, but had no significant effect on the time course of mepps in ColQ −/− muscle (from 3.57 ± 0.18 to 3.47 ± 0.07 ms in four experiments; P > 0.2). Together, these toxicological and physiological tests support the idea that ColQ −/− mutants lack functional synaptic AChE. We used sucrose density gradient centrifugation to assess the molecular forms of AChE present in ColQ −/− muscles. Globular forms are extracted into detergent-containing buffers at low ionic strength, whereas asymmetric (collagen-tailed forms) are extracted only at high ionic strength . Therefore, we first homogenized muscles with detergent-containing LS buffers, then reincubated the pellet with HS buffers to selectively solubilize asymmetric forms. Control ( ColQ + / + and ColQ +/− ) muscles contain three asymmetric forms, corresponding to a single helical trimer of ColQ chains attached to one, two, or three tetramers of catalytic subunits . In contrast, no asymmetric forms were detectable in ColQ −/− muscle . Because some asymmetric AChE is difficult to solubilize from tissue , we also assayed the pellet remaining after HS and detergent extraction. AChE activity in insoluble fractions was 14% of that in the HS extract for ColQ +/+ muscles ( n = 4), and 30% of that in the HS extract for ColQ +/− muscles. However, AChE activity was undetectable in the insoluble fraction from ColQ −/− muscles. Similar results were obtained with muscles from neonates (P0) and at P20. Thus, all asymmetric AChE in muscle requires ColQ for its assembly and/or accumulation. Surprisingly, loss of AChE in ColQ −/− mice was not limited to the asymmetric forms. At birth, three major peaks of AChE activity were distinguishable in detergent extracts of control muscle, corresponding to amphiphilic monomers and dimers (G 1 a and G 2 a ) and nonamphiphilic tetramers . G 1 a and G 2 a were retained in neonatal ColQ −/− muscles, but the G 4 na peak was almost entirely absent . In addition, the pool of globular tetramers extracted by HS was absent from ColQ −/− neonates . At P20, however, amphiphilic and nonamphiphilic tetramers (G 4 a and G 4 na ) as well as G 1 a and G 2 a forms were present in ColQ −/− muscles (data not shown). Thus, ColQ-independent mechanisms for assembly or retention of G 4 do exist (see Discussion). BuChE is homologous to AChE, hydrolyzes acetylcholine, and is present at the neuromuscular junction . Like AChE, BuChE catalytic monomers assemble into both globular and asymmetric multimers, and the asymmetric multimers bear a collagenase-sensitive subunit . To determine whether ColQ serves as the collagen tail for BuChE, we assayed sucrose density gradients in the presence of a selective inhibitor of AChE, BW284c51. Asymmetric and globular forms of BuChE were detectable in control muscle . As reported previously, S values of BuChE differed from those of AChE , providing assurance that the activity seen in the presence of BW284c51 did not reflect incomplete inhibition of AChE. Moreover, activity was inhibited by the selective inhibitor of BuChE, iso-OMPA (data not shown). Globular isoforms of BuChE were retained in ColQ −/− mutants, but no asymmetric BuChE was detectable . Thus, the ColQ gene appears to encode the collagenous tails of both AChE and BuChE. Lacking antibodies to BuChE, we used the histochemical stain of Karnovsky and Roots to localize BuChE in control and mutant muscles. This method relies on hydrolysis of the acetylcholine analogue, acetylthiocholine, which is cleaved by both AChE and BuChE. At P20, reaction product was readily detectable at ColQ −/− synaptic sites, although levels were substantially reduced compared with controls . The AChE inhibitor BW284c51 had no effect on the staining of ColQ −/− synaptic sites, but decreased staining in controls to approximately the level seen in mutants . This result provides independent evidence that AChE is depleted from ColQ −/− neuromuscular junctions. In contrast, the BuChE inhibitor iso-OMPA decreased staining of control synaptic sites only slightly, but completely abolished staining of ColQ −/− synaptic sites . Thus, the synaptic acetylcholine-hydrolyzing activity in mutants is largely or entirely BuChE. Reaction product was undetectable in controls even after prolonged incubation when a mixture of BW284c51 plus iso-OMPA was added with the substrate, indicating that AChE and BuChE together account for most or all of the ACh-hydrolyzing activity at control synapses . To obtain information on the molecular forms and cellular localization of synaptic BuChE, we treated cross-sections in various ways then stained them in the presence of BW284c51. In both control and ColQ −/− muscle, reaction product was tightly associated with synaptic sites . Pretreatment of sections with either collagenase, which releases matrix-associated enzyme, or with the detergent Triton X-100, which releases membrane bound or intracellular enzyme, reduced staining of synaptic sites in control muscles . Combined treatment with collagenase and Triton X-100 removed all detectable BuChE . Thus, both matrix- and membrane-associated BuChE are present at control synapses. In ColQ −/− muscle, collagenase treatment had no effect on the level of BuChE but detergent treatment abolished all staining , confirming the absence of matrix-associated BuChE and showing that residual activity is membrane-associated. Denervation, which causes loss of nerve terminals and rapid remodeling of terminal Schwann cells, led to complete loss of this membrane-associated BuChE from ColQ −/− synapses but had little effect on controls . Together, these results suggest that both membrane- and matrix-associated forms of BuChE are normally concentrated at the neuromuscular junction. The BuChE of the synaptic cleft is predominantly matrix-associated, asymmetric, and requires ColQ for assembly or localization. Membrane-associated BuChE that persists in the absence of ColQ, in contrast, is likely to be associated with nerve terminals or terminal Schwann cells, although we cannot exclude the possibility that it is postsynaptic. Although the synapse-associated BuChE in ColQ −/− muscles did not appear to be localized to the synaptic cleft, it might have been near enough to hydrolyze released acetylcholine. We tested this possibility by determining whether iso-OMPA affected the decay of mepps recorded from ColQ −/− neuromuscular junctions. No significant effect was seen (τ = 2.70 ± 0.15 ms in normal saline and 2.98 ± 0.49 ms in the presence of 10 μM iso-OMPA; n = 4; P > 0.2). Therefore, although there is synapse-associated BuChE in ColQ −/− mice, it does not appear to provide a mechanism to compensate for loss of AChE. To assess the size and shape of neuromuscular junctions in ColQ −/− muscle, thick longitudinal sections were stained with rhodamine-α-bungarotoxin. In controls, the AChR-rich membrane is a roughly circular plaque at birth, then passes through a perforated-plaque stage and eventually matures into a pretzel-like array of distinct AChR-rich branches by P20 . The precise geometry of branches is unique at each synaptic site, but the general pattern is stereotyped. In mutant homozygotes, in contrast, synaptic geometry was variable. Some synaptic sites were smaller than controls but roughly normal in appearance , some retained the immature appearance characteristic of ∼1-wk-old controls , and some appeared fragmented . Approximately 40% of the neuromuscular junctions were normal in geometry, another 40% were fragmented, and the remaining 20% were immature. Electron microscopy revealed two structural abnormalities at synaptic sites in ColQ −/− muscles. First, the cytoplasm beneath the postsynaptic membrane was often riddled with holes, consistent with a localized degenerative response . Such local degeneration has been observed after acute inhibition of AChE , and is believed to result from entry of Ca 2+ through excessively activated AChRs . Consistent with this interpretation, signs of necrosis were seldom observed in nonsynaptic regions of ColQ −/− muscle, and no signs of muscle fiber degeneration and regeneration (such as central nuclei, which are diagnostic of immature or regenerated fibers) were detectable. Second, nerve terminals were sometimes partially enwrapped by processes of Schwann cells . Such enwrapment has been seen in several pathological situations, and can occur after muscle damage, or as a direct consequence of abnormalities in the synaptic cleft . Because Schwann cell processes are impermeable to neurotransmitter, their intrusion into the synaptic cleft might represent one adaptive mechanism by which ColQ −/− synapses limit activation of AChRs. Interestingly, the incidence of subsynaptic necrosis and Schwann cell enwrapment of nerve terminals varied as a function of age. At P20, nearly two-thirds of synaptic sites encountered (80/125) showed clear signs of necrosis. By 6 mo of age, however, <5% of synaptic profiles (2/71) showed necrosis, and many were indistinguishable from controls . This difference raises the possibility that damage occurring early is repaired and that compensatory mechanisms, such as Schwann cell enwrapment, prevent damage from recurring. Indeed, the incidence of nerve terminals partially or completely enwrapped by Schwann cell processes increased somewhat, from one-third (42/125) at P20 to over one-half (39/71) at 6 mo of age. This difference suggests either that Schwann cell enwrapment is progressive, or that individuals with more complete enwrapment are more likely to survive. Necrosis and Schwann cell wrapping were seen in only 1 and 4 of 83 control endplates, respectively. Finally, we stained sections of muscle from ColQ −/− mice and littermate controls with a panel of antibodies to proteins present at synaptic sites. These included the transmembrane proteins α-sarcoglycan and β-dystroglycan, which are present throughout the muscle fiber but concentrated in the postsynaptic membrane; rapsyn and utrophin, which are selectively associated with the subsynaptic cytoplasm; and agrin, which is concentrated in synaptic basal lamina . The distribution of all of these markers was qualitatively similar in control and homozygous mutant mice . We also stained sections with the lectin VVA-B4. VVA-B4 binds to glycoconjugates that terminate with a β- N -acetylgalactosaminyl residue and selectively stains the synaptic cleft at the neuromuscular junction . We showed previously that VVA-B4 binds to asymmetric but not globular forms of AChE in muscle, but presented indirect evidence that other VVA-B4–binding proteins were also present in the synaptic cleft . VVA-B4 stained synaptic sites nearly as intensely in ColQ −/− muscle as in control muscle , providing direct evidence for the existence of additional synaptic VVA-B4–binding moieties. Asymmetric forms of AChE are most abundant in skeletal muscle, but are also present in several other tissues . To determine whether asymmetric AChE in nonmuscle tissues requires ColQ for assembly, we analyzed HS extracts of brain and heart. Low levels of asymmetric AChE were detectable in both tissues from control mice but in neither tissue from ColQ −/− mice . We also made a preliminary survey of tissue structure in the major organs of ColQ −/− mice, but found no abnormalities. Previous biochemical studies have shown that the ColQ protein is a collagenous subunit of asymmetric AChE, and suggested that it is critical for accumulation of AChE in the synaptic cleft of the neuromuscular junction. Analysis of ColQ −/− mutant mice has now allowed us to ascertain, in situ, the roles that ColQ plays in the assembly and anchoring of AChE and in the development and function of the neuromuscular junction. Specifically, we have been able to answer the seven questions posed in the Introduction. First, no asymmetric AChE is present in muscles that lack ColQ, presumably because the ColQ protein forms the collagen tail that endows asymmetric forms with their distinguishing feature. This result was expected but not inevitable. For example, ColQ might have been only one of multiple collagenous subunits, a possibility suggested by immunological studies showing a structural change in the collagen tail during posthatching development in birds . Alternatively, a synaptic collagen that normally plays other roles might have been capable of compensating for lack of ColQ in mutants. Second, muscles from newborn ColQ −/− mice lack not only all asymmetric AChE, but also the nonamphiphilic tetrameric form of AChE, G 4 na . This observation was unexpected because the G 4 na molecules have been assumed to be homotetramers of AChE T catalytic subunits. Possible explanations for this result include the following. G 4 na molecules may be derived from collagen-tailed molecules by proteolysis of the collagen tail, either in vivo or after extraction. Assembly of the tetramer might require transient association of AChE T with ColQ. Assembly might require an alternatively spliced product of the ColQ gene that contains the amino-terminal PRAD domain, but not the collagenous domain. Such alternatively spliced products do in fact occur , but have not yet been characterized. Some of the G 4 na may comprise tetramers associated with a single full-length collagen chain rather than a triple helix; such molecules sediment around 10S (Krejci, E., unpublished results). In any event, muscles of ColQ −/− mice did contain both amphiphilic and nonamphiphilic tetramers (G 4 a and G 4 na ) at P20. These tetramers might have been assembled without any organizer or might contain an alternate organizing subunit such as the still incompletely characterized P subunit . Thus, although the PRAD of ColQ can induce tetramerization of AChE T , and may do so in vivo at P0, there are clearly additional PRAD-independent mechanisms for forming tetramers. Third, no detectable AChE is present at synaptic sites in ColQ −/− muscles. Histochemical, immunochemical, toxicological, and electrophysiological assays all gave similar results in this regard. The lack of synaptic AChE could reflect loss of collagen-tailed AChE and/or loss of G 4 forms. We favor the interpretation that all synaptic AChE is collagen-tailed because all of the histochemically detectable AChE can be removed by collagenase treatment of sections (data not shown) or tissue . In addition, all globular forms (G 1 a , G 2 a , G 4 a , and G 4 na ) are present in ColQ −/− mice at P20, yet synaptic AChE is absent at all ages tested. Globular AChE is associated with isolated basal lamina from frog muscles , but this may result from the alterations produced in the experimental system of muscle and nerve degeneration. Fourth, ColQ is required for assembly or accumulation of asymmetric BuChE as well as AChE. In birds, collagen-tailed molecules containing both AChE and BChE subunits have been detected in muscles of hatchlings , suggesting that both enzymes use a common tail. Likewise, Krejci et al. showed that antibodies to ColQ interact with both AChE and BuChE as assessed by sucrose density centrifugations. Taken together with these biochemical data, our genetic results strongly support the conclusion that the ColQ gene produces the collagen tails for both AChE and BuChE. Fifth, the function of the neuromuscular junction is impaired in the absence of ColQ. However, the defects are not as severe as might be expected, considering the dramatic effects of acutely administering extremely selective blockers of peripheral AChE such as fasciculin. In particular, movements of ColQ −/− mice are relatively normal, while fasciculin-treated normal mice are paralyzed. Moreover, the postsynaptic necrosis observed in ColQ −/− weanlings, a likely result of excessive calcium influx through AChRs , is largely absent from those mutants that survive to later ages. These observations suggest that neuromuscular junctions compensate for the absence of AChE. Compensation does not appear to involve persistence or upregulation of BuChE: asymmetric BuChE is lost in the mutant and the BuChE inhibitor, iso-OMPA, does not affect the time course of mepps. Instead, ultrastructural studies suggest a simple structural mechanism to limit the amount of neurotransmitter that reaches the postsynaptic membrane: invasion of the synaptic cleft by Schwann cell processes at ColQ −/− synapses. Enwrapment of nerve terminals by Schwann cell processes is, in fact, actively regulated by another component of synaptic basal lamina, laminin-11 . ColQ might exert a similar effect. In addition, compensatory changes in quantal size and quantal content may occur; such homeostatic changes have been documented in response to several chemical or genetic perturbations of the efficacy of neuromuscular transmission in both vertebrates and invertebrates . Currently, we are using electrophysiological methods to seek such alterations in ColQ −/− mice, with the aim of learning how the synapse combines structural and physiological compensations to adapt to the challenge posed by AChE deficiency. Sixth, initial formation of the neuromuscular junction proceeds in the absence of ColQ and AChE. AChE promotes the extension of neurites from cultured motoneurons by a noncatalytic mechanism , and overexpression of AChE in transgenic frogs and mice perturbs formation of neuromuscular junctions in vivo . Our results imply that the developmental roles of ColQ and AChE at the synapse are either subtle or masked by compensatory mechanisms. However, it remains possible that expression of AChE at other sites is crucial for synaptogenesis. Moreover, at later stages, there are clear defects in the maturation of ColQ −/− neuromuscular junctions: some remain immature in geometry and others appear fragmented. These abnormalities could reflect a direct requirement for AChE or ColQ in synaptic maturation or maintenance, but might also be secondary consequences of subsynaptic necrosis or alterations in activity levels. Further experiments will be required to distinguish these alternatives. Seventh, ColQ is required for assembly or accumulation of asymmetric AChE in all tissues tested, but tissues other than skeletal muscle are not obviously defective in structure or function. However, it is premature to conclude that ColQ plays no roles in these tissues. For example, asymmetric AChE is concentrated in synaptic layers of the retina ; analyses of these synapses have not yet been undertaken. In addition, it is tempting to speculate that in AChE-poor tissues, ColQ or other PRAD-containing products of the ColQ gene serve to anchor proteins other than AChE to membranes or matrix. Identification of such proteins will guide the search for subtle phenotypes in ColQ −/− mice. Finally, it is interesting to note the parallels between ColQ −/− mice and humans with AChE-deficiency syndrome (CMS-Ic). This syndrome was described by Engel et al. over 20 years ago, and was reported just this year to result from defects in the human COLQ gene . Some patients appear to be protein nulls, while others have mutations in the carboxy terminus of ColQ that permit formation of asymmetric AChE but prevent its stable association with the basal lamina of the synaptic cleft. All of the COLQ patients are severely myasthenic, as are the ColQ −/− mice, making the mice a valid model for assessing pathogenic mechanisms and therapeutic interventions. | Study | biomedical | en | 0.999996 |
10102933 | A family of integral membrane proteins, called connexins, forms specialized cell–cell channels that serve as intercellular pathways of chemical and electrical information transmission. The cell–cell channel is assembled from the docking of two hemichannels (i.e., connexin hexamers) located in the plasma membrane of adjacent cells. This mechanism of cell–cell channel assembly suggests that there may be pools of hemichannels in the plasma membrane of participating cells. Although the physiological significance of undocked hemichannels in cell permeability and homeostasis is unclear, evidence has accumulated that hemichannels can function independently as non- or cation-selective, large conductance ion channels whose activity is dependent on the extracellular calcium concentration and intracellular pH . Cloning of the connexins, together with the introduction of efficient expression systems such as Xenopus laevis oocytes, opened new experimental avenues to study connexin hemichannels. While the expression of all connexins in oocytes leads to the formation of functional cell–cell channels by paired oocytes , only a few connexins form functional hemichannels in the plasma membrane . Here, we have combined electron microscopic and electrophysiological methods to study mouse connexin50 (Cx50) 1 expressed in Xenopus oocytes. Our approach combines hemichannel functional assays and the determination of the number of hemichannels in the plasma membrane of the same cell to assess the contribution of these channels to cell conductance. We show that expression of Cx50 in oocytes led to the insertion of functional hemichannels into the plasma membrane. Measurements of the total number of hemichannels in the plasma membrane and the Cx50 current in the same oocytes suggested that hemichannels rarely open at physiological calcium concentrations. When the density of hemichannels in the plasma membrane reached 300–400/μm 2 , they formed complete channels (dodecamers) and assembled into gap junctional plaques. The morphological studies further allowed the identification of hemichannels in vesicles shuttled to and from the plasma membrane. In the cytoplasm, hemichannels were present in small (∼0.1-μm diameter) “coated” and in larger (0.2–0.5-μm diameter) vesicles. The coated vesicles fused with the plasma membrane, whereas the larger vesicles originated from endocytosis of the plasma membrane. Trafficking of Cx50 hemichannels took place without changing the area of the plasma membrane, indicating that the rate of vesicles insertion into the plasma membrane was balanced by the rate of vesicle retrieval. Mature Xenopus laevis oocytes were injected with 50 nl distilled water or 50 nl 1 μg/μl mouse Cx50 cRNA , and incubated at 18°C in Barth's medium (mM: 88 NaCl, 1 KCl, 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 0.82 MgSO 4 , 2 NaHCO 3 , and 10 HEPES/Tris, pH 7.4) containing 0.1 mg/ml gentamicin . Electrophysiological experiments were performed with a two-electrode voltage-clamp technique . Oocytes were placed in a NaCl buffer (mM: 100 NaCl, 2 KCl, 1 CaCl 2 , 1 MgCl 2 , and 10 HEPES/Tris, pH 7.5) and the membrane potential was held at −50 mV. The whole-cell Cx50 currents were induced by varying the concentration of external calcium ([Ca 2+ ] o ). The Ca 2+ concentration was adjusted as desired by adding appropriate amounts of Ca 2+ and EGTA . To obtain current–voltage (I–V) relations, the pulse protocol consisted of 1,000-ms voltage steps from a holding potential (V h ) of −50 mV to a series of test voltages from +50 to −150 mV in 20-mV decrements (pCLAMP; Axon Instruments ). The voltage clamp had a settling time of 0.2–0.8 ms. Currents were low-pass filtered at 500 Hz, and sampled at 1 ms per point. Membrane capacitance measurements were used to estimate the total surface area of the plasma membrane . The capacitance was determined by applying 10–20-mV test pulses (V t ) of 30–100-ms duration from the holding potential (−50 mV). At each test voltage, the charge ( Q ) was obtained by integration of the capacitive transient. In both control and Cx50-expressing oocytes, capacitive transients showed a single time constant (∼1 ms), and Q was a linear function of the voltage step (V t − V h ). The slope of the Q versus (V t − V h ) relation was the membrane capacitance C m . In oocytes expressing Cx50, capacitance measurements were made at 5 mM [Ca 2+ ] o to avoid hemichannel activation. Membrane conductance was obtained as either the slope of the steady state I–V relationship or, where indicated, as a chord conductance . The reversal potential (V rev ) was determined by subtracting the endogenous currents at 5 mM [Ca 2+ ] o . All experiments were performed at 21 ± 1°C. After functional measurements, oocytes were fixed at room temperature and prepared for morphological studies . The fixative solution contained 3–3.5% glutaraldehyde in 0.2 M Na-cacodylate (pH 7.35). Oocytes were postfixed in 1% OsO 4 in 0.2 M Na-cacodylate buffer for 90 min at room temperature. They were washed in 0.1 M Na-acetate buffer (pH 5.0) and block-stained in 0.5% uranyl acetate in 0.1 M Na-acetate buffer (pH 5.0) overnight at 4°C. They were dehydrated in ethanol, passed through propylene oxide, and embedded in Polybed 812. Sections were cut in a Sorval MT5000 ultramicrotome ( Dupont ), collected on single-hole formvar-coated grids, and stained with uranyl acetate and lead . Control oocytes and oocytes injected with cRNA were incubated for 1 h in Barth's solution containing 0.5–1.5 mg/ml of peroxidase ( Sigma Chemical Co. ), and were then fixed in 3–3.5% glutaraldehyde in 0.2 M Na-cacodylate (pH 7.35) for 2 h at room temperature. They were washed and incubated in 0.1 mg/ml diaminobenzidine in 0.1 M Tris/HCl (pH 7.5) and H 2 O 2 , and then prepared for thin sectioning. Fixed oocytes were infiltrated with 25% glycerol in 0.2 M Na-cacodylate (pH 7.35) for 1 h (21 ± 1°C). Each oocyte was cut into six to eight small pieces (∼1 mm 3 ) to obtain replicas from different regions of the surface of the oocyte. The pieces were placed on Balzers specimen holders with the external surface (i.e., the vitelline membrane) facing upward. This orientation yielded extensive P face areas of the plasma membrane, which allowed us to determine the density and size of the newly inserted particles . The specimens were frozen by immersion in liquid propane maintained just above freezing in a liquid nitrogen bath. The frozen specimens were transferred to a Balzers 400 K freeze-fracture-etch apparatus, fractured at either −150° or −120°C at ∼10 −7 mbar of partial pressure. The fractured surfaces were coated with platinum at 80°C and carbon at 90°C. Replication at 80°C decreased the length of the shadow of the particle, which greatly facilitated the density quantification and the measurement of the particle diameter. The specimens were coated with 0.5% collodion in amyl acetate to avoid fragmentation of the replica, and cleaned in a solution of bleach to dissolve the organic material. The replicas were washed in distilled water and deposited on formvar-coated, single-hole copper grids. The collodion was removed by immersion in amyl acetate . The large area of the oocyte plasma membrane (∼3 × 10 7 μm 2 ) introduced sampling difficulties since only a small percentage of the area (6–8 × 10 2 μm 2 ) was imaged and used for the determination of the particle density. To address this, P faces collected from three to four replicas from different regions of the plasma membrane of each oocyte were imaged at 25,000×. Approximately 100 negatives were collected from each replica. From this pool, ∼20 negatives per replica free of optical aberrations and containing an area of 1–2 μm 2 of uninterrupted P fracture face were printed and used for quantification (Table I ). Altogether, the data were obtained by studying over 1,000 negatives from replicas of the fracture faces of the plasma membrane of control oocytes and oocytes injected with Cx50 cRNA. In replicas obtained from different poles of Cx50-expressing oocytes (vegetal versus animal), no differences were observed in the Cx50 hemichannel density or the fractional area covered by gap junction plaques. The images were enlarged to 75,000×, digitized, and analyzed using IMAGE software (National Institutes of Health). In each image, we measured the area of the P face and the total number of particles contained in that area. The particle density in the microvilli plasma membrane was quantified independently to ensure that it was similar to that of other regions of the plasma membrane. The diameter of the intramembrane particles was measured perpendicular to the direction of the shadow directly from negatives using a Profile Projector (6C; Nikon Inc. ) at a final magnification of 250,000× . The number of hemichannels in gap junction plaques ( n gj ) was estimated using n gj = K A t 2 d , where K is the fractional area covered by plaques per 100 μm 2 of plasma membrane, A t is the total area of the plasma membrane calculated from the capacitance, d is the mean density of particles in plaques (9,196 ± 694/ μm 2 ), and 2 because each gap-junction particle is composed of two hemichannels. The density of endogenous P face particles in the plasma membrane of control oocytes was 196 ± 9 particles/μm 2 (mean ± SEM, n = 8, n refers to the number of P face regions from which the particles were sampled). To detect the expression of Cx50, the particle density had to increase at least to 250/μm 2 ; i.e., 54 particles/μm 2 (two SDs) above the mean particle density of control oocytes, which represents a confidence level of ∼98%. The highest density at which each particle could be resolved without interference from the shadows of neighboring particles was ∼10,000 particles/μm 2 (approached in gap junctional plaques). The electrophysiological properties of Xenopus laevis oocytes injected with Cx50 cRNA were dependent on [Ca 2+ ] o . When measured 3 d after injection, in 1 mM external Ca 2+ , the resting membrane potential of Cx50-expressing oocytes was significantly different from that of control oocytes. Control oocytes had a resting potential of –50 ± 5 mV (mean ± SEM, n = 5) and a membrane conductance of 1.7 ± 0.2 μS ( n = 5), while the resting potential of oocytes injected with Cx50 cRNA was −33 ± 2 mV ( n = 7), and the membrane conductance was 7.2 ± 0.8 μS ( n = 7). When the [Ca 2+ ] o was elevated to 5 mM, the resting membrane potential and conductance of Cx50-expressing oocytes were restored to those of the controls (not shown). The dependence of the membrane conductance of oocytes expressing Cx50 on [Ca 2+ ] o was studied under voltage-clamp conditions. Fig. 1 A (top) shows a continuous current record from an oocyte expressing Cx50 and maintained at a holding potential (V h ) of −50 mV. Reduction in [Ca 2+ ] o from 1 mM to 10 μM induced an inward current of ∼1,450 nA. This inward current was reversibly blocked (∼65%) by intracellular acidification by addition of acetate (50 mM equimolar Na-acetate replacement for NaCl, pH 7.5) to the bathing medium. The current returned to baseline after restoration of [Ca 2+ ] o to 1 mM. The response of control oocytes to the same experimental protocol is illustrated in Fig. 1 A (bottom). Reduction of [Ca 2+ ] o from 1 mM to 10 μM induced a small outward current (9 ± 3 nA, n = 27), which was insensitive to intracellular acidification. This experiment was repeated in more than 70 control oocytes and in none was there a Ca 2+ -sensitive current as that detected in the Cx50-expressing cells. This suggests the absence of endogenous connexin hemichannels in our control oocytes. 2 A feature of gap junction channels is their inhibition by octanol, heptanol, and halothane . We tested the effect of 1-octanol on the currents activated by low [Ca 2+ ] o . Fig. 1 B shows a current trace from an oocyte held at −50 mV. Reduction in [Ca 2+ ] o to 10 μM led to an inward current of ∼900 nA that was reversibly inhibited by ∼95% by addition of 1 mM octanol to the bathing medium. Octanol had no effect on the holding current of control oocytes (not shown). We further studied the Ca 2+ sensitivity of the inward current at −50 mV . From 10 −2 to 10 −6 M [Ca 2+ ] o , there were small incremental increases in the holding current; however, when [Ca 2+ ] o was lowered to 10 −7 M, the current increased by ∼3.3-fold . In Ca 2+ -free solutions (10 mM EGTA and 0 Ca 2+ ), the Cx50 currents were too large for reliable control of the membrane potential. In control oocytes, when the membrane potential was stepped from the holding (−50 mV) to test voltages, the capacitive transient decayed to a steady state with a time constant of ∼1 ms . This relaxation was independent of the [Ca 2+ ] o (data not shown). The steady state I–V relations in 5 mM or 10 μM external Ca 2+ were similar . Membrane conductances in 5 mM and 10 μM external Ca 2+ in the oocyte in Fig. 2 A were 1.2 ± 0.1 and 1.3 ± 0.1 μS. The current response of Cx50-expressing oocytes after step changes in membrane potential depended on [Ca 2+ ] o . In 5 mM [Ca 2+ ] o , the currents were not appreciably different from control oocytes, and the conductance was 2.4 ± 0.1 μS. In 10 μM [Ca 2+ ] o , large Cx50 currents resulted that, after the initial capacitive transient, decayed slowly to a steady state . At steady state, the inactivation was dependent on [Ca 2+ ] o (not shown) and the magnitude of the voltage step. It was most prominent at low [Ca 2+ ] o and at the largest voltage steps . At the smallest voltage steps, there was no apparent inactivation as the relaxation of the current transient was not appreciably different from that of the capacitive transient, but at the largest voltage steps (±100 mV), the time constant of inactivation ranged from 220–300 ms. The Cx50 I–V relationships were measured at 10 (I initial ) and 980 (I steady-state ) ms after the onset of the voltage pulse . I initial was proportional to the size of the voltage step, resulting in a near linear I–V relationship. I steady-state deviated from linearity and at the extreme voltage steps (−150 to −90 mV and +30 to +50 mV), the conductance was negative. Membrane conductance was 41 ± 1 μS for I initial (−150 to +50 mV) and 35 ± 1 μS for I steady-state (−70 to +30 mV). Both I initial and I steady-state reversed at the same membrane potential (−25 mV), indicating that they were carried by the same ionic species. Equimolar (100 mM) replacement of external Na + with choline shifted V rev for both currents to more negative values (approximately −35 mV). V rev was not significantly affected by replacement of external Cl − with gluconate or Mes (not shown) or by variations in [Ca 2+ ] o . Therefore, in oocytes expressing Cx50, the functional and pharmacological properties of the current induced at low [Ca 2+ ] o were consistent with the expression of a functional connexin hemichannel and, henceforth, this current will be referred to as I Cx50 . I Cx50 was used as a functional assay of the presence of Cx50 in the plasma membrane. The magnitude of I Cx50 was dependent on the length of time after cRNA injection . I Cx50 appeared as early as ∼3 h after injection (data not shown), increased steadily up to the third day and, thereafter, decreased . Although this time course was very reproducible, the magnitude of I Cx50 was dependent on the amount of cRNA injected and the frog from which the oocytes were harvested. Control oocytes (V h = −50 mV) did not exhibit a Ca 2+ -sensitive current over this time course . The membrane capacitance ( C m ) of oocytes injected with Cx50 cRNA was not different from that of control oocytes up to 5 d (300–600 nF). In both groups of oocytes, C m remained unchanged up to the third day and decreased thereafter . Thus, Cx50 expression occurred without altering the plasma membrane area. We focused on the organization of the plasma membrane and the neighboring protoplasmic region (“cortical” region). The plasma membrane of control oocytes contained numerous microvilli, folds, and invaginations that increased its surface area by approximately ninefold compared with a smooth spherical cell of similar diameter . The cytoplasm of the cortical region contained secretory granules closely associated with the plasma membrane , small (∼0.1 μm) and large (0.2–0.5 μm) diameter vesicles, mitochondria, and endoplasmic reticulum. Oocytes expressing high levels of Cx50, as assessed from the magnitude of I Cx50 and the density of the plasma membrane particles (see below), exhibited gap junctions that appeared as pentalamellar structures 16–18 nm in overall thickness . The gap junctions were either continuous (“reflective”) or discontinuous (“annular”) with the plasma membrane. Reflective gap junctions were formed by the association of the plasma membrane with a microvillus or a fold , or in membrane invaginations . Annular gap junctions appeared as vesicles in the cytoplasm and most likely originated from invaginations of the plasma membrane, which later pinched off into the cytoplasm . Freeze-fracture micrographs of the reflective and annular gap junctions revealed that the P face of the plasma membrane contained plaques of particles, and the complementary E (external) face contained the corresponding pits . The plaques contained Cx50 hemichannels assembled into gap junctions. The height of the fracture step separating the junctional membranes was 6–7 nm, and the extracellular space between the two membranes was 1–2 nm, which is consistent with that found in gap junctions. Annular gap junctions appeared as concave and convex surfaces with plaques of particles and complementary pits also separated by fracture steps of small height . Gap junctions were not observed in control oocytes , in oocytes expressing low levels of Cx50 (<300 P face particles/μm 2 ), or in oocytes over- expressing other heterologous membrane proteins (in the absence of Cx50), such as the Na + /glucose cotransporter (SGLT1), occludin, aquaporin-0, aquaporin-1, or Shaker K + channel. We have observed gap junction formation in oocytes expressing connexin46 (Zampighi, unpublished observations). In thin sections, oocytes expressing high levels of Cx50 were identified by the presence of gap junctions, which appeared 48–72 h after cRNA injection . In freeze fracture, expression of Cx50 was evident ∼24 h after cRNA injection by the appearance of a distinct population of intramembrane particles in the P face of the plasma membrane . The newly inserted (Cx50) particles increased the density of particles in the P face, and the Cx50 particle density was correlated with the magnitude of I Cx50 . To characterize the particles that appear after the expression of Cx50, we compared the size and shape of the P face particles in control oocytes and those expressing Cx50 using frequency histograms. In the P fracture face of control oocytes, ∼93% of the endogenous proteins had a mean diameter of ∼7.5 nm, and the rest had a mean diameter of ∼11 nm . The density of the P face particle was 196 ± 9/μm 2 (mean ± SEM, n = 8). The complementary E face of the plasma membrane of control oocytes contained ∼13-nm particles at a density approximately four times higher than the particle density in the P face (886 ± 36 particles/μm 2 ). The density of the E face particles did not change after Cx50 expression. The size (diameter) frequency histograms of the particles in oocytes expressing Cx50 exhibited a population of 9.0 ± 0.4 nm (mean ± SEM, n = 544) diameter particles , in addition to the endogenous integral membrane proteins . The newly inserted particles exhibited a symmetrical cross-sectional geometry that contrasted sharply with the size and shape of the endogenous particles . When the density of the newly inserted particles reached 300–400/ μm 2 , they formed gap junction plaques (see below). Size frequency histograms also showed that the particle in the gap junction plaques exhibited the same size and shape as the newly inserted particle population . Therefore, expression of Cx50 induced a population of intramembrane particles of distinct size and shape (hexamers), which assembled into plaques of complete channels (dodecamers) at a critical density. We previously characterized the population of newly inserted P face particles induced by the expression of other heterologous membrane proteins, such as aquaporin-0, aquaporin-1, SGLT1, and opsin. Particle size analysis showed that each protein was represented by a particle with a distinct size and shape whose cross-sectional area predicts the number of alpha helices in the transmembrane domain . An analysis of the cross-sectional area of the particle induced after the expression of Cx50 predicted that each Cx50 particle could accommodate 24 ± 3 α helices . Therefore, based on the size and cross-sectional area of the particles, and their ability to form gap junctions, we conclude that Cx50 is inserted into the plasma membrane as hemichannels (hexamers). The density of Cx50 hemichannels in the P face (excluding those in gap junction plaques) was examined at various times after Cx50 cRNA injection . To relate the density measurements to functional ones, I Cx50 (at −50 mV and 10 μM [Ca 2+ ] o ) and C m were also measured in the same oocytes (Table II ). Quantification of the P face particles in oocytes examined at different times after injection showed that the density of hemichannels was proportional to I Cx50 (Table II ). At ∼6 h after injection, the current was small (approximately −10 nA) and the density of particles did not increase significantly above that of endogenous particles measured in control oocytes . At 24 h, the magnitude of I Cx50 increased to 415 nA and the hemichannel density to ∼200/μm 2 above background . At 48 h, I Cx50 and hemichannel density increased approximately twofold . At this level of Cx50 expression, hemichannels assembled into gap junctions plaques of P face particles . I Cx50 and the density of Cx50 hemichannels peaked at 72 h ; however, they decreased by ∼25% with longer incubation times (Table II ). The initial rate of Cx50 expression (Table III ) indicates that hemichannels were inserted into the plasma membrane at a rate of 80,000 copies/s. The area of the oocyte plasma membrane was estimated from whole-cell capacitance measurements (assuming 1 μF/cm 2 ; Table II ), which together with I Cx50 (at −50 mV and 10 μM Ca 2+ ) obtained from the same oocyte, allowed for the calculation of the specific membrane conductance (in picosiemens per square micrometer). The specific membrane conductance plotted as a function of the Cx50 hemichannel density (per square micrometer) from the same oocytes, yielded a linear relationship, the slope (2.7 × 10 −3 pS) of which corresponded to the product of the open probability ( P o ) and the single channel conductance (γ) of the Cx50 hemichannel . Gap junction plaques were first observed 48 h after cRNA injection when Cx50 hemichannel density reached 300–400/μm 2 and, thereafter, occupied a fairly constant area of the plasma membrane (1.3–1.7%). At peak expression, the area covered by plaques contained 8.2–9.2 × 10 9 hemichannels; 35–40% of the total number of Cx50 hemichannels in the plasma membrane (∼2.6 × 10 10 ; Table III ). We took advantage of the distinct size and shape of the Cx50 hemichannel particles, as well as their ability to form gap junctions in single oocytes, to identify the vesicles involved in the insertion/retrieval pathway to and from the plasma membrane. Two types of vesicles within the cytoplasm contained hemichannels . One was located close to the plasma membrane and to organelles such as the Golgi complex, the endoplasmic reticulum, and lysosomes . These vesicles measured ∼0.1 μm in diameter and contained slender projections, spaced ∼20-nm apart, which protruded from the vesicle surface . Some coated vesicles were fused with the plasma membrane through a narrow neck 25–30 nm in length and a diameter ranging from 20 to 40 nm . In freeze fracture, these vesicles appeared as concave and convex surfaces and, depending on the expression level, contained 5–40 Cx50 hemichannels . These vesicles also contained the 13-nm endogenous particles found on the E face. The other type of vesicle with Cx50 hemichannels exhibited a larger diameter (0.2–0.5 μm) and an irregular shape . Markers of the extracellular space, such as peroxidase, showed electron-dense reaction products in the lumen of these vesicles . Therefore, the larger vesicles were either connected to the extracellular space or originated from invaginations of the plasma membrane that ultimately pinched off into the cytoplasm . In freeze fracture, the large vesicles were characterized by the presence of Cx50 hemichannels; i.e., 9-nm particles in the P face and the complementary pits in the E face . Some large vesicles contained hemichannels at a density similar to that in the gap junction plaques described in the plasma membrane , and most likely correspond to the annular gap junction seen in thin sections . Other large vesicles contained hemichannels at lower densities or in small clusters located in highly curved regions of the vesicle . These large vesicles may correspond to early endosomes or the trans-Golgi network. The E fracture face of the vesicles provided another important clue regarding their origin. These faces contained the complementary pits of the Cx50 hemichannels at a density similar to plaques , or arranged in small clusters . In addition to the hemichannels, the E face of the large vesicles also contained large 13-nm-diameter particles . These E face particles correspond to a population of endogenous proteins in the plasma membrane of control oocytes . Therefore, the vesicles identified in this study contained Cx50 hemichannels intermingled with endogenous proteins of the oocyte plasma membrane, suggesting that trafficking of endogenous and heterologous proteins uses a common pathway. Injection of Cx50 cRNA into oocytes leads to the appearance of a new population of particles (Cx50 hemichannels) in the plasma membrane, whose density is directly correlated with a whole-cell Ca 2+ -sensitive current (I Cx50 ). Several observations suggest that the newly inserted particles represent Cx50 hemichannels, and that I Cx50 is in fact mediated by Cx50 hemichannels. First, the newly inserted particles exhibited a circular geometry with a diameter of 6.5 ± 0.5 nm (after correcting for the thickness of the platinum-carbon film; 2.4 nm) . The cross-sectional area, 34 ± 4 nm 2 (πr 2 ), can accommodate 24 ± 3 α helices . Both the particle diameter and the cross-sectional area observed here are consistent with the reported values from two-dimensional crystals of gap junction membrane channels , and ordered arrays of hemichannels . The size and shape of the Cx50 particle suggest a hexameric hemichannel composed of four α helices per subunit. Second, at a density of 300–400/μm 2 , Cx50 particles formed reflective gap junctions in the plasma membrane of single oocytes. This property has not been observed for endogenous proteins in control oocytes, or in oocytes expressing a wide variety of other heterologous membrane proteins. Third, oocytes expressing Cx50 demonstrated a current (I Cx50 ) that was activated at low [Ca 2+ ] o . The properties of I Cx50 suggest that it is mediated by a nonselective cation channel. I Cx50 could be blocked by octanol or intracellular acidification, other characteristics of gap-junction channels and hemichannels . Finally, at low [Ca 2+ ] o and large voltage steps, there was a time-dependent inactivation of Cx50 macroscopic currents, such that the steady state I–V relationship displayed regions of negative slope. Similar voltage-dependent characteristics have been observed for other connexin hemichannels . Altogether, the functional, pharmacological, and morphological characteristics observed in Cx50-expressing oocytes point to the expression of connexin hemichannels. A unique aspect of this study was that the total number of hemichannels inserted into the plasma membrane was estimated in the same oocytes in which I Cx50 was measured. This allowed us to estimate the unitary properties of Cx50 hemichannels. The increase in membrane conductance due to expression of Cx50 hemichannels (G Cx50 ) is proportional to the number of hemichannels ( n ), the single hemichannel conductance (γ), and the channel open probability ( P o ) ( G Cx50 = P o γ n ). Combined freeze-fracture electron microscopy and electrophysiological measurements were used to determine the total number of hemichannels ( n ) and the membrane conductance ( G Cx50 ) in the same oocytes. At −50 mV and 10 μM [Ca 2+ ] o , P o γ for Cx50 hemichannels was −2.7 × 10 −3 pS . Using 30 pS for Cx50 single channel conductance (obtained under physiological extracellular and intracellular Na + and K + concentrations and symmetrical 100 nm [Ca 2+ ]; Eskandari, S., and D.D.F. Loo, manuscript in preparation), we estimate that at 10 μM [Ca 2+ ] o , Cx50 P o is −9 × 10 −5 ; i.e., only ∼1 in 10,000 hemichannels is open at any given time. In light of the observed Ca 2+ sensitivity of G Cx50 , we predict that Cx50 hemichannels would rarely open at physiological [Ca 2+ ] o (1–2 mM). These data suggest that the whole-cell conductance induced at peak expression by lowering [Ca 2+ ] o to 10 μM (46 μS; Table II ) results from the opening of ∼1.5 × 10 6 hemichannels (γ = 30 pS), representing ∼0.009% of the total number of single hemichannels in the plasma membrane (1.7 × 10 10 ; Table III ). This suggests that, in the oocyte, there is one conducting hemichannel every ∼20 μm 2 of the plasma membrane. At a more physiological [Ca 2+ ] o (1–2 mM), G Cx50 decreases by ≥20-fold, which would correspond to one open hemichannel for every ∼400 μm 2 of the plasma membrane. If these findings in oocytes are extrapolated to a smaller cell, such as a liver hepatocyte, and a density of 500 hemichannels/μm 2 is assumed, we estimate that there would be two conducting hemichannels per cell. Therefore, our data suggest that at physiological [Ca 2+ ] o , Cx50 hemichannels play a small role in single cell permeability and homeostasis, and the potential physiological significance of this hemichannel would be confined to large cells possessing a high density of hemichannels, conditions which favor channel opening (e.g., low [Ca 2+ ] o ), or pathophysiological states that may result in apoptosis. After Cx50 cRNA injection, the total number of hemichannels in the plasma membrane reached a steady state (2.3–2.6 × 10 10 ) at 72 h (Table II ), which correlated with the electrophysiological assay of hemichannel function . Based on the number of hemichannels in the oocyte plasma membrane 24 h after cRNA injection (Table II ), the initial insertion rate of Cx50 into the plasma membrane was ∼80,000 hemichannels/s. The observation that Cx50 hemichannels were colocalized in the same vesicles with endogenous proteins of the oocyte suggests that Cx50 over-expression does not alter the oocyte's constitutive translation and trafficking machinery. Assuming that insertion is via fusion of the small coated vesicles (0.1 μm diameter) with the plasma membrane, and that each vesicle contains 5–40 Cx50 hemichannels, we predict that Cx50 over-expression should increase the plasma membrane by 63–503 μm 2 /s (5.4–43.4 × 10 6 μm 2 in 24 h). In spite of such a sizable rate of insertion, there was no increase in the area of the plasma membrane . Thus, exocytosis was balanced by endocytosis. It is interesting to note that at peak Cx50 expression, these rates of exocytosis and endocytosis would lead to the replacement of the entire plasma membrane of the oocyte in ∼24 h! In contrast, expression of other heterologous membrane proteins such as SGLT1 and aquaporin-0 increased the area of the plasma membrane of oocytes up to fourfold. In the case of SGLT1, the increase in the plasma membrane area occurs concurrently with an increase in the number of transporters in the plasma membrane, and the rate of SGLT1 insertion is comparable to that observed here . Therefore, it appears that the delivery of some endogenous membrane proteins, as well as heterologous proteins such as SGLT1 and Cx50, to the plasma membrane involves the same mechanism. The difference in the trafficking of the heterologous membrane proteins appears to involve the retrieval mechanism. In the case of Cx50, endocytosis of large Cx50-containing vesicles balances the rate of small vesicle insertion. In oocytes expressing SGLT1, however, this pathway does not balance the rate of vesicle exocytosis, resulting in a continuous increase in the area of the plasma membrane. Cx50 was inserted into the plasma membrane as functional hemichannels. No gap junctions were observed until the density of hemichannels in the plasma membrane reached a threshold of 300–400/μm 2 . Therefore, the plaques of channels were assembled from the recruitment of the Cx50 hemichannels in the plasma membrane. Once maximal expression levels were reached, the fraction of hemichannels in nonjunctional and junctional membrane regions remained relatively constant (Table III ), indicating that there was a steady state between the insertion of hemichannels into the plasma membrane and their entry into junctional plaques. Retrieval occurred through endocytosis of both hemichannels and complete channels assembled in junctional plaques (see below). Assuming a steady state at maximal expression levels (72 h), we estimate that the half-life of a single hemichannel (junctional and nonjunctional combined) in the plasma membrane was ∼3.7 d. This is longer than the metabolic half-life estimated for other connexins in other expression systems . We identified Cx50 hemichannels in small (∼100 nm) and large (0.2–0.5 μm) diameter vesicles. We interpret these vesicles as components of the trafficking machinery involved in Cx50 insertion into and retrieval from the plasma membrane. Three observations suggested that the larger vesicle originates from plasma membrane invaginations and is a component of the retrieval pathway. First, when the hemichannel density reached 300–400 μm 2 in the plasma membrane, the invaginations assembled gap junctions that pinched off and appeared as annular gap junctions in the cytoplasm . Second, the extracellular marker, peroxidase, labeled the lumen of the large, but not the small, vesicles. Third, these larger vesicles contained hemichannels intermingled with an endogenous plasma membrane protein of the oocyte (i.e., the 13-nm particle in the E face). Altogether, these results suggest that Cx50 hemichannel retrieval shares a pathway with endogenous proteins of the plasma membrane of the oocyte. In this scheme, the large vesicles represent early endosomes formed by endocytosis of large invaginations of the plasma membrane. The origin and fate of the smaller coated vesicles containing the hemichannels were difficult to determine. One possibility is that they insert the hemichannels into the plasma membrane, as their lumen was not observed to be labeled with peroxidase. This interpretation is likely because these vesicles were observed to be associated with Golgi-resembling flattened membrane sacs. An alternative possibility is that the coated vesicle is involved in the endocytosis of hemichannels only (not gap junctions) from the plasma membrane, and subsequently delivers them to an early endosomal compartment. While additional information will be required to distinguish between the two hypotheses, our identification of vesicles containing hemichannels represents a necessary first step for a more accurate description of the trafficking of heterologous proteins to and from the plasma membrane of oocytes. In summary, our results led to the hypothesis that Cx50 hemichannel insertion into the plasma membrane was via exocytosis of small coated vesicles . This pathway was shared by both endogenous and Cx50 proteins. This process steadily increased the density of Cx50 hemichannels in the plasma membrane until a steady state level was reached at 72 h. Retrieval of Cx50 hemichannels from the plasma membrane occurred via endocytosis of large vesicles. At low density (<300/μm 2 ), retrieval of hemichannels started in invaginations of the plasma membrane that pinched off and formed large cytoplasmic vesicles. This pathway was also shared by both endogenous and Cx50 proteins. At higher densities (>300/μm 2 ), the large invaginations formed reflective gap junctions that pinched off and appeared as annular gap junctions in the cytoplasm. The principal characteristic was that, despite the ongoing insertion and retrieval processes, the area of the plasma membrane remained constant. | Study | biomedical | en | 0.999995 |
10102934 | Mechanosensitive (MS) 1 channels or stretch-sensitive channels, discovered in chick skeletal muscle cells are likely candidates for the role of primary mechanoreceptors in unicellular and multicellular organisms. In the hair cells responsible for hearing and balance in vertebrates, MS channels are implicated in generation of primary potentials associated with the hair bundle displacement . In nonsensory cells, MS channels have been shown to mediate mechanical stress-induced changes in membrane permeability to monovalent ions and Ca 2+ , potentially triggering cascades of second messenger signaling. In bacteria that live in a rapidly changing environment, mechanosensitive channels mediate permeation of small osmolytes from the cytoplasm through the periplasm to the extracellular space, potentially permitting a rapid regulation of turgor pressure . MS channels are identified by their activities, not sequence similarities. Given the variety of observed ionic selectivities and gating properties, there is little evidence that they form a family by sequence similarity . In eucaryotes, MS channel activity appears to require coupling to the cytoskeleton and/or the extracellular matrix, probably for the transmission of force . In contrast, bacterial MscL is functional in lipid bilayers . Although some MS channels have been characterized biophysically, very little is known about their molecular structure. An extensive genetic dissection of the nematode Caenorhabditis elegans revealed ∼20 genes involved in touch sensation . Two of them are possible candidates for MS channel subunits, featuring close sequence similarity to the ENaC, the amiloride-sensitive Na + channel . Other touch genes code for cytoskeletal or extracellular matrix components, presumably those elements necessary for the efficient transmission of forces to the channels. Unfortunately, there are only two descriptions of electrophysiological evidence for the mechanosensitive role of any of these genes . As an alternative to the complexity of metazoa, MS channels in microorganisms are biochemically and genetically accessible . Patch-clamp studies on native and reconstituted Escherichia coli membranes revealed three types of mechanosensitive channel (Msc) activities: MscL, MscS, and MscM . Using a variety of chromatographic techniques followed by reconstitution and patch recording of channels in liposomes, MscL, the most conductive of MS channels in E. coli was identified as an ∼17-kD protein, and the corresponding mscL gene was then cloned . Biochemical studies have shown that MscL resides in the inner membrane of E. coli . Each MscL subunit is a 15-kD protein with two putative transmembrane domains and a high α-helical content . Whereas the functional channel complex was proposed to be a homohexamer , a recent crystallographic study indicates a pentameric structure for the closed state of the channel MscL can be activated by 70– 180-mmHg pressure gradients across patches of bacterial spheroplasts, and purified MscL channels reconstituted into phospholipid liposomes produced similar currents. This indicates that MscL can be gated directly by tension transmitted via the lipid bilayer alone. In vivo, the channel may be opened by osmotic gradients of 200–700 mOsm . MscL-like channels are found in several groups of eubacteria , and there is increasing evidence that MscL plays the role of a “safety valve” in prokaryotes, releasing small osmolytes, there- by reducing the turgor pressure and the chance of cell lysis. In the present work, we quantitatively evaluate the energetic parameters for MscL gating in reconstituted liposomes using a similar approach as described by Opsahl and Webb . We used high-resolution video microscopy to measure the curvature of patches at different activating pressures, permitting us to calculate the absolute tension. Kinetic and thermodynamic analysis of the channel allows us to calculate the free energy differences between states and their tension dependence . Close examination of the single channel conductance has identified at least four open conductance classes. Analysis of transition rate constants, using a simple linear Markov model, has permitted identification of the tension dependence of each rate constant between the closed and open conductance classes. This work represents the first calibration of a cloned, biological mechanosensitive ion channel. The procedure for MscL purification using a 6His-tag has been described previously . Briefly, a tag of six sequential histidines was added to the COOH terminus of MscL by a two-step PCR amplification and the extended gene was cloned into the pB10a vector . The PB104 cells expressing MscL-6His were French-pressed and the total membrane fraction was isolated. The MscL-6His protein was extracted from membranes by solubilization in 3% β-octylglucoside and purified in one step using a Ni-NTA column (QIAGEN Inc.) as described . Azolectin (Soybean lecithin, type II; Sigma Chemical Co. ), a lipid component of proteoliposomes, was partially purified from oxidized and lyso forms using chloroform/water separation. Briefly, 500 mg of azolectin beads were washed five times with 5–7 ml of acetone (electron microscopy grade), with gentle swirling after each change. After the last wash, the residual acetone was removed by vacuum (20 min) and the beads were dissolved in 5 ml of chloroform. The solution was placed in a thick-walled glass tube with a Teflon stopper, overlaid with 5 ml of water, and shaken vigorously. The cream-colored mixture was separated by 2 h centrifugation in a swinging-bucket rotor at 13,000 rpm at 15°C. The lower chloroform portion was retrieved by a long-needle glass syringe, placed in an airtight Teflon-capped vial, and could be stored at −20°C under nitrogen for ∼1 mo. MscL-6His was reconstituted into azolectin liposomes by dialyzing the β-octylglucoside-solubilized mixture of a protein-to-lipid ratio of 1:500 to 1:2,000 . In special cases when we needed to record single MscL currents for kinetic analysis, the protein-to-lipid ratio was lowered to ∼1:10,000. Proteoliposomes were subjected to a dehydration–rehydration cycle on glass slides and the resultant multilayer aggregates were placed in the recording buffer (see below) for 30–60 min before the patch-clamp experiment. Large, and apparently unilamellar, blisters formed on the surface of multilayer liposomes were examined as excised inside-out patches. Borosilicate glass pipettes with 1–2-μm bore diameter were used in all experiments. The pipette pulling protocol was adjusted to form long-tipped pipettes with an almost cylindrical, 10–20-μm– thick by 100-μm–long region before the tip. This narrow taper was useful for observation of liposome patches that tend to creep up the pipette under pressure gradients. To compensate for the headstage tilt and make the observed part of the pipette nearly parallel to the focal plane, pipette tips were bent as described previously . All recordings were performed in a symmetrical buffer containing 200 M KCl, 40 mM MgCl 2 , and 10 mM HEPES, pH 7.2. Pressure gradients were delivered by either a pneumatic screw-driven syringe and monitored by an electronic pressure transducer or with a laboratory-built hydraulic pressure servo . Patch currents were recorded at hyperpolarizing voltages (+20 mV in the pipette) using an Axopatch 200 ( Axon Instruments ) and stored on a PCM tape (48 kHz sampling rate; Instrutech Corp .). The second data channel on the recorder was allocated to recording pressure. To determine P o as a function of pressure, the data were analyzed using PCLAMP6. P o was calculated as the mean patch conductance G P divided by n · G MscL , where G MscL is the maximal conductance of a single MscL channel (typically 3.5–3.7 nS) and n is the number of channels in the patch. n was estimated either by measuring the current at saturating pressures or, with less precision, by using the average number of channels per patch in a given proteoliposome preparation determined in separate experiments. When n was >10, the patch conductance, G P , was corrected to account for the series pipette resistance ( R S ≅ 1.5–2 MΩ) by the equation, G P = I /(V − IR S ), where V and I are the transmembrane voltage and current, respectively. We made >200 attempts to record complete activation curves for MscL in different settings and, of these, fifteen were considered extensive enough to warrant analysis. They compose the data presented below. Experiments were usually terminated by lysis of the patch. For the multistate analysis, we used the QuB program suite ( www.qub.buffalo.edu ). To determine the rate constants between states, the digitized data was first idealized using SKM, a Hidden Markov algorithm . The “events list” outputs from SKM were then grouped as a global collection of data sets over defined tension. This tension series was fit to a kinetic model using MIL, a maximum likelihood interval analysis program that permits data to be fit across independent variables and corrects for missed events . Since we determined there were five states (four subconducting and one shut state), the number of possible kinetic connections was extremely large (728 models). We were able to exhaustively search all models and connectivities using the program MSEARCH, which employs MIL to rank all models, based on the likelihood calculated for each model. For all nonlooping models, the linear sequential model gave the greatest likelihood, and therefore was used for the analysis: C1–S2–S3–S4–O5, where C, S, and O refer to the closed, subconductance, and open states, respectively. To determine how the single channel MscL conductance depends on the bath conductivity, we recorded currents from reconstituted channels in baths containing 10 mM HEPES, pH 7.2, and 40 mM MgCl 2 , with KCl concentrations varied between 0.1 and 2 M. The specific conductivity of each buffer was measured directly using a YSI 34 Conductance Meter equipped with a dip-type glass cell with platinized platinum-iridium electrodes (1.0 cm cell constant; Yellow Springs Instrument Co.). Single-channel currents were recorded at +20 mV (pipette voltage), and the unitary current of the fully open state was determined using FETCHAN's ‘Measure' function (PCLAMP suite; Axon Instruments ). Patches were imaged using an inverted microscope (Axiovert; Carl Zeiss, Inc. ) equipped for Differential Interference Contrast with a CCD camera as initially described by Sokabe et al. . The digitized images were analyzed with an algorithm written in JAVA by Akinlaja that solved for the radius of curvature. To avoid confusion within equations, T will reference tension, while temperature T will only appear as a term with the Boltzmann constant; i.e., k B T . Previous patch-clamp experiments revealed that activation of MscL requires pressures close to the lytic tension of the unmodified bilayer . This proximity made it difficult to obtain many data sets with saturating responses. Typically, activation in spheroplast patches required 70–200 mmHg, while liposome patches needed 40–150 mmHg. Patches formed with large-diameter pipettes activated at lower pressures, suggesting that the actual parameter that drives MscL gating is tension, not pressure. Prolonged exposure of patches to high pressures often caused lysis. This imposed strict requirements on the stability of the bilayer in which the channels were reconstituted. A partially purified crude phospholipid fraction from soybean (azolectin) gave us acceptable stability and reproducibility of results. Under a small pressure gradient, liposome patches, lacking cellular components, are spherical caps and are large enough to be viewed by conventional optical microscopy. Experiments were considered successful if we were able to obtain enough points to fit the partial activation curve with P o > 0.3 with images of the patch of sufficient quality to calculate the curvature. As the data below will show, MscL is not a binary channel, but has multiple conducting states. However, some of the essential features of the gating process can be gained from considering the simpler two-state model, a model that would correspond to the data viewed at low bandwidth. This analysis is based on setting a threshold for being open at half the fully open state amplitude. Previous data indicated that mean current MscL pressure–activation curves can be well fit with single component Boltzmann distribution. Our initial experiments were designed to determine the midpoint ( T 1/2 ) and the maximum slope of this distribution, the two key parameters of the dose–response curve. Only one parameter, T 1/2 , actually requires the measurement of patch curvature. An accurate measure of the slope can be obtained from P o ( P ) curves measured “blindly” (i.e., without geometric measurements), provided that the radius of curvature is independent of the pressure (see below). Fig. 1 shows a typical trace from a patch containing ∼100 MscL channels in response to a stepwise increase of pressure gradient P (bottom). The MscL current activates at ∼40 mmHg and increases with P in a nonlinear manner. Note that in the beginning of the trace the variance of the current (amplitude of fluctuations around the mean level) increases with each step, reaches a maximum, and finally decreases during the last pressure step before the patch ruptures. The maximum of the current variance indicates the point at which P o = 0.5; thus the half-maximal pressure P 1/2 is between 48 and 52 mmHg for this particular recording . The R s -corrected dose–response curve derived from this trace is shown in Fig. 1 , inset. Fig. 2 shows the geometry of the same patch at pressure gradients of 0, 20, 44, and 52 mmHg. In the absence of suction, the patch is essentially flat, subjected to the “resting” tension arising from membrane adhesion to the glass surface . At low suction, the patch appears as a spherical cap, with a progressively increasing curvature . At higher suction, the curvature saturates because the membrane is nearly inextensible (see calculation below). At higher suctions, the patch may creep up the pipette . This creep does not affect the geometry significantly as long as the pipette taper is small. As seen in Fig. 1 , MscL channels are only active for P > 40 mmHg. This is the range of pressures where the patch curvature reaches saturation so that one measurement of the curvature was adequate to calculate the tension for all pressures where MscL is active. The procedure of fitting the patch curvature is depicted in Fig. 3 . We have been able to obtain partial activation curves from four independent patches, in which we were also able to measure the radius of curvature, r . The membrane tension was calculated for every pressure according to Laplace's law, T = p × r /2, and the P o data were plotted against T as shown in Fig. 4 . The patches were fit as a group to a single Boltzmann function with T 1/2 = 11.8 ± 0.8 dyn/cm and a maximal slope of 0.61 ± 0.17 dyn/cm per e-fold change of P o / P c . More precise and statistically reliable estimates for the slope of P o ( T ) curves were obtained from the analysis of additional P o ( P ) experiments done without measurement of the patch curvature, but aligned to T 1/2 = 11.81 dyn/cm. Given that the patch curvature does not change significantly in the range of pressures where MscL is active , the two scales, T and P , are equivalent with T 1/2 = r × P 1/2 /2. Measuring P o ( P ) curves alone is much easier than the simultaneous geometric measurement. 11 complete P o ( P ) curves obtained from three independent MscL preparations were fit independently with a Boltzmann curve. P 1/2 was determined individually for each curve, and then rescaled to the same midpoint, T 1/2 = 11.81 dyn/cm. As shown in Fig. 5 , the curves display a remarkable consistency and, when fitted together, indicate the slope factor of 0.63 ± 0.08 dyn/cm. If interpreted in the framework of a two-state Boltzmann model with the change of in-plane area being the dominant energy term, T Δ A , we have: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{o}=1/[1+exp({\Delta}E-T{\Delta}A)/k_{B}T].\end{equation*}\end{document} Fitting to Eq. 1 gave Δ E = 18.61 k B T (46.3 kJ/mol) for the free energy of the closed-to-open transition in the absence of stress, and Δ A = 6.52 nm 2 for the in-plane change in area between closed and open ( k B T is Boltzmann's constant × the absolute temperature = 4.04 × 10 −14 erg at room temperature). In a more specific representation of the two-state model, we can write the forward and backward rates as: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{on}=k_{0}\;exp(-a{\times}T)\;and\end{equation*}\end{document} 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{off}=k_{0}\;exp(-b{\times}T),\end{equation*}\end{document} where k 0 is a scaling factor incorporating T 1/2 and a and b are functions of the energies between the energy wells and the top of the barrier separating the states . For example, a = Δ A c / k B T , where Δ A c is the increase of in-plane area between the closed state and the top of the energy barrier. The units of a are inverse tension (cm 2 /erg = cm/dyne). Since the probability of being open, P o = k 12 /( k 12 + k 21 ), the Boltzmann equation assumes the form P o / P c = exp[( a − b ) T ]. Fig. 7 represents typical single-channel traces illustrating the MscL kinetics at different tensions (A) and semi-logarithmic tension dependencies of rate constants for opening and closing transitions ( k on and k off ) derived from these traces according to a two-state model (B). The dependence of ln( P o / P c ) for the same patch illustrates the rate at which the equilibrium occupancies of open and closed states change with tension. The tension dependence of the rate of opening ( k on ) is about equal to that of ln( P o / P c ). The tension dependence of the rate of closing is four times less. This suggests that the shortening of the closed state makes a larger contribution to the change of P o than the lengthening of the open state. From the tension dependence of the rate constants, we estimate that A c − A b is ≈ 4.42 nm 2 (equivalent to a disk of radius 1.19 nm) and A o − A b ≈1.37 nm 2 , (equivalent to a disk of radius 0.7 nm). The total area change, based on transition rates between closed and open, is therefore 5.8 nm 2 , as compared with 6.5 nm 2 calculated from P o . As shown below, the channel is not a two-state system and therefore a discrepancy may be expected. The above analysis was done as though the channel were a two-state system with rate constants calculated from dwell times generated according to a half-amplitude detection criterion . Closer examination of the data, however, shows multiple conducting states . The conductances illustrated in Fig. 8 are shown in Table I , along with estimates of the pore radius calculated assuming a cylindrical pore 4.2-nm long . While the minimal reaction scheme for so many states would normally be difficult to determine reliably, exploration of the likelihood of all possible interconnections indicated that the simple sequential model (shown below) was the best fit for all nonlooping models (Table II ). The rate constants for all patches are plotted as a function of tension and are shown in Fig. 9 . The straight lines are nonlinear regressions to a simple exponential of k = k 0 exp(α T ) with the residuals weighted by the inverse of the variance provided by MIL. The parameters of the regression are shown in Table III . Most striking is that only k 12 has a significant positive slope; all other rates are insensitive or have negative slopes. In the simple free energy model presented above, the tension sensitivity α can be identified with Δ A / k B T . One needs to bear in mind, however, that the sign of α will change with the direction of the reaction. A transition from a well to a barrier will produce the opposite change in area than moving from the barrier to the same well. To summarize the kinetics contained in Fig. 9 and Table III , we have plotted in Fig. 10 the energy profiles of the states and the changes of in-plane area. Fig. 10 shows again that the rate limiting step to opening channel is k 12 , for which the energy barrier is ∼38 k B T . At zero tension, the energy difference between the closed and any of the conducting states is >18 k B T , accounting for the fact that the channel is almost never open at rest. All states of conductance >S1 have about the same energy and are insensitive to tension. Applying tension lowers the energy of all the conducting states (energy wells) and barriers equally. The dimensional changes of the channel are shown in Fig. 10 , bottom, where we plotted a running sum of the area changes accompanying each transition. As expected from the tension sensitivity, the area changes associated with changing states are small except for the transition from closed to S1. The total change of in-plane area between the closed and open states is 5–6 nm 2 , as estimated from the two-state analysis. How well does the above analysis fit reasonable physical models of the channel? There are two types of information we can use to estimate the physical structure: the pore diameter and the channel protein properties. The channel conductance gives information about the aqueous pore, and the protein properties give estimates of the wall thickness. We measured the MscL full conductance state as a function of bath conductivity. As shown in Fig. 11 , the fully open single channel conductance was linear with the conductivity up to 2 M KCl. The absence of saturation and the lack of anion/cation selectivity is consistent with the representation of the open MscL as a wide aqueous pore. The conductance data were fit with the Hille equation describing conductance of a cylindrical pore , and the results are shown in Table IV . The first column corresponds to the assumed length of the channel. The calculated cross-sectional area suggests that the open MscL channel has a diameter of 2.7–3.6 nm, which is in good agreement with the data reported recently by Cruickshank et al. . The value for in-plane area expansion during gating estimated from the two-state analysis (above), Δ A = 6.5 nm 2 , is in the same range estimated for the pore cross-section, 5.8 > A > 9.7 nm 2 (see Table IV ). Having an estimate of the open channel pore diameter, we now need to estimate the wall thickness to obtain the outer diameter where the bilayer tension is applied. The channel is a pentamer, with each monomer containing two helical transmembrane segments . Given that the fully open channel will have a pore diameter of ∼4 nm, there are barely enough α helices to coat an aqueous pore of the estimated diameter if the helices make a close-contact circle around the pore. Using 1 nm as an estimate for helix diameter, the outer diameter of the open channel will be ∼6 nm. Alternatively, adding the change in radius of ∼0.5 nm (calculated from the change in area seen by gating) to the crystal radius of the closed channel predicts an outer diameter of open channel of 5.5 nm. The structure of the lower conductance states, however, is less well constrained since the in-plane area changes associated with gating do not match the changes in conductance. If internal pore reorganization is involved in setting the conductance of the closed and substates, we would predict each substate to exhibit a distinct dependency on the ionic composition of the bath. MscL, the first mechanosensitive channel for which some structural characteristics are available, now has quantitative parameters that can be used to judge the effects of mutagenesis and pharmacology. The ability to use a fluid bilayer for reconstitution eliminated the need to deal with the complex mechanics of heterogeneous biological membranes. The low bending moment of bilayers assured the membranes were spherical so that the pressure gradient could be simply translated into tension according to Laplace's law. Since bilayers have substantial resistance to area changes, there was little change in curvature once the membrane assumed a spherical shape. The image-fitting algorithm used to determine patch curvature was limited by the optical resolution of the microscope (∼0.35 μm), such that changes in the radius of curvature could not be reliably detected at the higher pressures. The expected changes can be predicted from an equation describing the stretch of an elastic membrane attached to the pipette walls : 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P=(4K_{A}/a)\;(T/K_{A})^{3/2}/(1+T/K_{A}),\end{equation*}\end{document} where K A is the area elastic constant of the membrane and a is the radius of the pipette where it meets the patch. Using Laplace's law, we can then solve for the radius of curvature r as a function of pressure. Inserting a few constants makes the behavior clear. Consider a soft membrane with K A = 100 dyn/cm and a patch with a = 2 μm. For P = 50 mmHg (1 mmHg = 1.33 × 10 3 dyn/ cm 2 ), r = 3.33 μm and for 100 mmHg, r = 2.76 μm, a change of 20%. For a stiffer membrane with K A = 500 dyn/cm, the same pressures give r = 5.44 and 4.38 μm, a change of 24%. Thus, in this pressure range, the tension is nearly proportional to the pressure. The set of data in Fig. 4 shows that the tension to activate MscL is near the lytic strength of the bilayer. The P o ( T ) curves have a midpoint of 11.8 dyn/cm, which exceeds the critical tension for mechanical breakdown of many phospholipid bilayers . This is also close to the lytic tension for bacterial membranes as suggested by frequent lysis of spheroplast patches at tensions that activate MscL. This proximity is consistent with the proposed “safety valve” function for MscL as a pore that can dissipate osmotic gradients when the membrane stability is in danger. In a cell of 1-μm diameter, the osmotic gradient required to create a tension of 12 dyn/cm is 20 mOsm, corresponding to a pressure of 360 mmHg. Soil bacteria may experience much larger osmotic stresses during rainfall. Knockouts of MscL have not shown osmotic fragility, however, and this is probably caused by the presence of other more sensitive mechanical channels, including MscS and MscM . MscL may become active and release larger osmolytes only at extremely strong downshocks, >200 mOsm, as indicated by osmotically-induced ATP efflux (Sukharev, unpublished observations). The nonsaturable character of MscL conductance and the absence of anion/cation selectivity strongly suggest a large water-filled pore. The specific conductivity of electrolyte inside the pore would then be similar to its bulk value and, therefore, the “macroscopic” equations deriving the channel conductance from its geometry and conductivity are reasonable . The length of the channel pore was estimated to be ≈4 nm from sieving data . The energetics of MscL gating suggests that this channel undergoes large changes in dimensions, accounting for its steep dose–response curve. MS channel gating is, by definition, a function of force . The simplest model for planar mechanical free energy is based on the notion that if the open channel occupies a larger area than the closed channel, then tension favors opening. This model was used in the energy diagram of Fig. 6 , although it should be pointed out that while the figure is drawn with finite widths for the wells and barriers, the mathematics assumes the barrier is a delta function and the wells are boxes. The dose–response curves in Fig. 5 were well fit by Eq. 1 with parameters Δ E = 18.6 k B T (46.3 kJ/mol) and Δ A = 6.5 nm 2 . Both numbers are large at the molecular scale and their combination makes the MscL dose– response curve steep. The large value of Δ E predicts that the open probability at low tension ( T < 1 dyn/cm) would be ∼10 −8 . Indeed, with no mechanical stimulus, the impedance of a patch with >200 MscLs is >1 GΩ— closed channels are not leaky and have almost no spontaneous activity. If MscL opened spontaneously, it would dissipate the proton gradient within ∼1 μs and disrupt cell energetics. Using the slope of the tension–sensitivity curve, we can compare MscL gating energetics with other channels. Alamethicin and the mechanically sensitive cation-selective channel found in chick skeletal muscle have similar sensitivities on the order of 3–8 dyn/cm per e-fold change in P o . The yeast and bacterial MS channels described here have much steeper slope sensitivities (0.05 and 0.63, respectively), indicative of large area changes between closed and open states. The midpoints of the activation curves were quite different, however, with the yeast MS channel reaching half max with only 0.7 dyn/cm tension as compared with MscL, which required 11.8 dyn/cm. The setpoints and sensitivities of the MS channels so far examined appeared to be tuned to respond to membrane tension in very different ways, perhaps reflecting the different physiological roles. The similarity between the size of the MscL pore and the change of in-plane area Δ A oc is striking, and the basic relationship is maintained in the multistate analysis, where the C–S1, instead of C–O, is rate limiting. These dimensional changes strongly suggest that the opening of the pore constitutes the major part of the entire protein complex expansion. When the rate constants of Table I are taken explicitly in terms of an Eyring model, differences in k 0 s reflect differences in the entropy of activation. In turn, the entropy is a measure of deformability, with narrow energy wells representing stiff conformations and wide energy wells representing soft conformations . The rate constant k 12 has the largest entropy change of all the rates, suggesting that the closed state is quite flexible relative to the excited state (the peak of the barrier between 1 and 2). If we examine the change of stiffness between states by calculating the entropy differences, ΔΔ S , the only significant change of stiffness is between the closed state and S1. Thus, the closed state seems to be the only one sufficiently soft to allow the available tension to do significant work on the channel. Do these results agree with structural information? Each MscL subunit (15 kD, 136 amino acids) spans the membrane twice with both termini intracellular. The two transmembrane domains are most likely α helical and oriented normal to the membrane as judged by circular dichroism . Although initial cross-linking experiments had suggested that the functional MscL complex is a homohexamer , more recent and extensive cross-linking studies and the crystal structure of the closed channel indicate a pentameric stoichiometry . Images of negatively stained two-dimensional crystals of tag-purified MscLs revealed hexagonal lattices of doughnut-shaped particles that were recognized as hexamers. The resolution of unsymmetrized projection maps achieved in this work does, however, seem to be insufficient to draw an unambiguous conclusion on the number of subunits in the complex. As discussed by Cruickshank et al. , twelve transmembrane helices would be just sufficient to line a pore ≈4 nm in diameter. The 30 amino acid periplasmic loop might be a part of the pore lining of the open channel, but is clearly within the cytoplasm in the closed channel . In examining the energetic model as illustrated in Fig. 10 , the question arises as to why the increase in conductance from S1 to O5 (presumably an increase in cross-sectional area of the channel) is not correlated with a significant increase in the tension sensitivity—we know that the conductance, and presumably pore diameter, is increasing. There are two types of explanations: (a) the in-plane area really doesn't increase very much, or (b) the free energy available from tension described as T Δ A is incomplete. Explanations for a could involve increases in conductance from shortening the pore, rather than increasing the cross-section. Alternatively, there may be more complicated conformational changes to the pore interior that affect ion transport. Explanations for b might involve components of free energy such as the line tension, a term proportional to the perimeter of the channel that has the opposite sign to T Δ A . Line tension plays a role in the stability of pores in lipid bilayers . Or, we could have stress-induced changes in channel (water?) entropy that would affect the free energy . Currently, we cannot separate these various components, but the relative simplicity of internal pore reorganization as a method to alter conductance has appeal from studies on mechanically insensitive channels. To account for the similarity of Δ A for the rate-limiting step and the pore diameter, the closed conformation would have to exclude most of the water from its interior (∼15–30 nm 3 ), presumably becoming some sort of compact, ion-impermeable, bundle. If the helices remain normal to the membrane, the pore cannot be closed by steric interactions unless the channel forms a close packed trimer. If the helices twist about the pore axis, the effects of pore diameter are minor as long as the pore diameter is comparable to the membrane thickness. If the helices rotate axially, forcing hydrophobic faces toward the pore, the channel might close using ordered water as the “gate” , but this seems unlikely given the large diameter of the structure. If the channel closed by using the extracellular domain as a gate, then the agreement of the pore cross-section with the Δ A co must be assumed coincidental. Regardless of details, there are large dimensional changes involved in opening MscL; changes much larger than proposed for other channels. Our analysis has focussed on state models because of the clear finite residence times in the different conducting states. However, the large and rapid dimensional changes associated with gating probably also involve inertial components that affect the current rise times. Since the propagation velocity of shear waves in lipids are on the order of 10 nm/ms , there may be useful physical details in the form of the transition currents, particularly those between C and S1. Recently, Gu et al. proposed an electromechanical model of MscL gating that involves the NH 2 -terminal domains as gates, pivoting under stress from a position parallel to the membrane to one normal to the plasmalemma. The model attempts to calculate the electrostatic force between specific charged residues located on the NH 2 - and COOH-terminal domains and those on the membrane-spanning helices. Membrane tension causes the extracellular end of the helices to tilt inward, changing the distances between the charges, lowering the force that causes the NH 2 -terminal domains to swing to a normal position with respect to the bilayer. However, the balance of electrostatic forces between charged residues was calculated without accounting for electrolyte screening, which dictates that in 0.2 M salt there will be almost no interaction between two unitary charges positioned 1-nm apart. Our experimental data show there is no significant change in the gating pattern in the range of salt concentration between 0.05 and 1 M. This practically excludes the role of long-range electrostatic interactions in MscL gating. MscL is also weakly voltage dependent, which is contrary to the model where an external field must strongly influence the distribution between open and closed states. The Gu et al. model also imposes strict constraints on the length and charge of the NH 2 -terminal domain. This region must be about the pore radius in length, and six of them must occlude the pore completely, as we know that closed MscL is absolutely nonleaky. This is difficult to satisfy and also contradicts the work of Blount et al. and Hase et al. , whose data show that the removal of 3, substitution of 8, or addition of 20 new residues to this domain doesn't significantly change the channel gating. The model assumes the closed channel is already in a fully patent configuration; i.e. the 4-nm pore is present through all of the open and most of the closed states. Although the model predicts a small increase in area during gating, the ∼6-nm 2 area increase we calculate (which accounts for the steepness of the dose–response curve) is much larger than predicted. Even if the channel operates through a combination of area changes that pull the gates to a lower energy state followed by the NH 2 -terminal swinging gates, our evidence for large area changes does not seem compatible with their model. However, the subconductance states could result from individual terminal domains partially interfering with permeation. As this paper was in the process of review, Dr. Doug Rees (California Institute of Technology, Pasadena, CA) kindly shared with us a preprint of the full x-ray structure of an MscL homolog and we felt that it was useful to make a first-order comparison with our results. The structure looks like two barrels in series—a wide one in the transmembrane portion and a narrow one in the intracellular compartment. The channel is a pentamer with each subunit having two alpha helical transmembrane domains tilted at ∼28° and an intracellular helical domain. The intracellular domains form a 3.5-nm continuation of the pore. The result is a channel ∼8.5 nm in length with a diameter that varies from ∼1.8 to 0.2 nm, the latter representing the gate in the closed channel. This putative gating region consists of a ring of hydrophobic residues located near the intracellular depth of the bilayer. The outer diameter of the transmembrane portion, where tension is applied, is ∼5 nm. The most striking contrast with our results (and other published results) comes from the expected channel conductance. Knowing the open channel conductance, and modeling it as a cylindrical pore, we can calculate that a channel 4 nm in length must have a diameter >3 nm to have a conductance of ∼3 nS (Table IV ). If the pore were opened to its maximal 1.8-nm diameter along its entire 8.5-nm length, the predicted conductance would be ∼0.5 nS instead of the calculated 3.2 nS. If, upon opening, the intracellular pore domain were assumed to unfold completely, the transmembrane length would be ∼5 nm and the conductance 0.85 nS. Since the simple cylindrical model assumes no interaction of ions with the channel, the ions are point charges, and there are no image forces, the conductance estimate should be a maximum. Furthermore, since the channel is nonselective between anions and cations and its conductance is exactly proportional to the solution conductance up to 2 M KCl, we cannot invoke local fixed charges as concentrators of ions to increase conductance. It would appear that opening of the channel must involve major alterations in conformation that both shorten and widen the channel. The constraints are clear if we suppose tension causes the channel to splay into a cone with the narrow end of the pore remaining at the observed 1.8-nm diameter (presumably with the narrow end extracellular). As with a cylindrical pore, the conductance of a tapered pore consists of two convergence resistances and the pore resistance given by R pore = ρ l /π r 1 × r 2 , where the r is the radii at each end, ρ is the solution resistivity, and l is the pore length. If the intracellular portion of the channel were folded out of the way against the bilayer so the pore was only 4-nm long, we would still have to expand the internal diameter to 100 nm to get the observed 3-nS conductance. Constraining even one end of the channel to 1.8-nm diameter strongly limits the possible pore conductance. However, it is perhaps not surprising that the pore dimensions of the closed channel differ greatly from the dimensions predicted for the open pore. Concerning the structural origin of the mechanical sensitivity, the tension sensitivity can be explained by a modest increase in the outer diameter of the transmembrane domain from ∼5.0 to 5.5 nm. If each of the 10 transmembrane helices were 12–13 nm in diameter and arranged in a close-packed ring normal to the membrane, the outer diameter would be 5–6 nm, in the range necessary to account for the mechanical sensitivity. If tension untwists the transmembrane helices to form a set of barrel staves perpendicular to the membrane, the cytoplasmic domains may peel away, shortening the length of the pore. The substates we observed may reflect such movements of the cytoplasmic helices. These domains, which are outside the bilayer, should not be strongly driven by membrane tension, making them compatible with the lack of tension sensitivity of the higher conductance substates. There are methodological questions to be resolved between the crystallography and the electrophysiology. The crystals were formed in solutions at pH 3.7 and were stabilized with glutaraldehyde as well as heavy metal compounds, including Gd +3 , which is a known blocker of the channel. These conditions may create structures different from the native state and physiological tests need to be made on channels treated this way. Nonetheless, it is a thrill to have a real structure to examine and we eagerly await a structure of the open channel. In summary, we have performed the first calibration of a biological mechanosensitive ion channel. These measurements place strong constraints on kinetic and structural models of MscL and related channels. | Study | biomedical | en | 0.999998 |
10102935 | The cystic fibrosis transmembrane conductance regulator (CFTR) 1 is a member of the ATP-binding cassette or traffic ATPase superfamily. Proteins in this superfamily harvest the energy from the hydrolysis of ATP to transport a variety of substrates across the cell membrane . Like other members in this family, CFTR is predicted to contain two membrane spanning domains, each composed of six putative membrane-spanning segments and two nucleotide binding domains (NBD1 and NBD2). However, unlike other members, CFTR has a regulatory domain containing multiple consensus sites for phosphorylation via PKA; functionally, CFTR itself is a plasma membrane chloride channel . It is thought that regulation of CFTR channel activity is through PKA-dependent phosphorylation of the regulatory (R) domain. Although a prerequisite for activation, PKA phosphorylation of the R domain in itself is not sufficient for opening of the channel. Once the R domain is phosphorylated, nucleotide interaction with the NBDs is coupled to the opening and closing (i.e., gating) of the channel. The NBDs contain a highly conserved region known as the Walker A and Walker B motifs. It is believed that this region forms a close association with the phosphates of bound nucleoside triphosphates . From the sequence of the NBDs, it is predicted that they not only bind, but hydrolyze nucleotides. Biochemical experiments using purified CFTR have demonstrated that the protein functions as an ATPase . Molecular modeling by sequence comparison and functional studies of CFTR have suggested that the function of NBDs (especially NBD2) in CFTR parallels that of a G protein . Furthermore, recent studies have shown that the purified NBDs can hydrolyze ATP and GTP . The functional implication of these biochemical results is that CFTR may use the free energy of ATP hydrolysis to drive conformational changes associated with gating transitions. Previous work on CFTR gating by Hwang et al. and Gunderson and Kopito has established that nucleotide binding and hydrolysis at the NBDs is important in channel gating. However, each group proposed a different role for the two NBDs in gating of the channel. Hwang et al. proposed that hydrolysis at NBD1 is coupled to channel opening, while subsequent binding at NBD2 stabilizes the open conformation; hydrolysis of ATP at NBD2 triggers channel closing. In contrast, Gunderson and Kopito proposed that nucleotide binding and hydrolysis at NBD1 is only a prerequisite for channel opening; subsequent nucleotide binding at NBD2 is actually responsible for channel opening and hydrolysis at this second ATP binding site is responsible for channel closure. Both models are supported by the observations that the nonhydrolyzable ATP analogues, such as AMP-PNP and ATPγS, fail to open CFTR channels, but dramatically increase the open time in the presence of ATP . The fact that CFTR mutants with impaired nucleotide hydrolysis at NBD2 present a prolonged channel open time also supports these models. We studied gating of CFTR in excised inside-out patches from NIH3T3 cells stably transfected with wild-type (wt) or K1250A-CFTR. In wt-CFTR, both the mean open and closed times depend on [ATP]. This concentration dependence of the mean open time is more prominent in K1250A-CFTR, a mutant CFTR of which the conserved lysine residue in the Walker A motif of NBD2 is converted to alanine. Our kinetic data are consistent with a cyclic gating scheme that violates microscopic reversibility due to an input of energy from ATP hydrolysis. Both wt and K1250A (lysine to alanine mutation) CFTR channels were stably expressed in NIH3T3 cells . NIH3T3-K1250A cells stably expressing K1250A-CFTR were established using the retroviral vector pLJ (a generous gift from Dr. Mitchell Drumm, Case Western Reserve University, Cleveland, OH). Both cell lines were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine and 10% calf serum. For patch-clamp recording, cells were passaged and grown on small, sterile glass chips in 35-mm tissue culture dishes. Before recording, glass chips were transferred to a continuously perfused chamber located on the stage of an inverted microscope ( Olympus Corp. ). Patch-clamp pipette electrodes were made using a two-stage vertical puller (Narishige). The pipette tips were fire-polished with a homemade microforge to ∼1 μM external diameter, resulting in a pipette resistance of 3–6 MΩ in the bath solution. CFTR channel currents were recorded at room temperature with an EPC-9 patch-clamp amplifier (HEKA Electronik), filtered at 100 Hz with a built-in three-pole Bessel filter (Frequency Devices Inc.), and stored on videotape. Unless otherwise mentioned, data were subsequently played back at a refiltered frequency of 25 Hz with an eight-pole Bessel filter and captured onto a hard disk at a sampling rate of 50 Hz. Pipette potential was held at 50 mV in reference to the bath. Downward deflections represent channel openings. The pipette solution contained (mM): 140 N -methyl- d -glucamine chloride (NMDG-Cl), 2 MgCl 2 , 5 CaCl 2 , and 10 HEPES, pH 7.4 with NMDG. Cells were perfused with (mM): 150 NaCl, 2 MgCl 2 , 1 EGTA, 5 glucose, and 5 HEPES, pH 7.4 with 1 N NaOH. In excised inside-out patch mode, the superfusion solution contained (mM): 150 NMDG-Cl, 10 EGTA, 10 HEPES, 8 Tris, and 2 MgCl 2 , pH 7.4 with NMDG. Immediately after excision, patch pipettes were moved to a small homemade chamber where a complete solution change could be accomplished within 3–5 s. MgATP, salts, and buffers were purchased from Sigma Chemical Co. The catalytic subunit of PKA was purchased from Promega Corp. AMP-PNP was purchased from Calbiochem Corp. All nucleotides were dissolved in the NMDG-Cl solution used for excised studies at a stock concentration of 250 mM. When millimolar ATP was used in the superfusion solution, noticeable acidification occurred, so the pH of superfusion solutions were readjusted to 7.4 with NMDG after addition of ATP. The mean current amplitude of the macroscopic CFTR channel current was estimated by averaging the current over a 20–60-s stretch with Igor software (Wavemetrics). Curve fits of the time courses for deactivation by washout of AMP-PNP and ATP from patches containing wt-CFTR or the time courses for deactivation by washout of ATP from patches containing K1250A-CFTR were obtained by using the Igor software. Our goal is to understand gating transitions of CFTR related to ATP hydrolysis, which likely occurs on a time scale in the range of hundreds of milliseconds to seconds . However, short-lived transitions of tens of milliseconds or less are commonly observed in CFTR . These “flickers” are more prevalent in cell-attached patches , are voltage- dependent , and may be due to blockade of the channel by anions on the cytoplasmic side . To eliminate these flickers from our analysis, we filtered our signals at a relatively low cutoff frequency (−3 dB) of 25 Hz using an eight-pole Bessel filter. In addition, open events are defined as intervals separated by closings of 80 ms or greater. We found that using a cutoff time of <80 ms results in estimates of gating transitions that are too fast to be consistent with ATP hydrolysis-dependent gating (see discussion ). Our 25 Hz filter setting results in a 10–90% rise time of 12 ms; therefore, ATP-dependent gating transitions are likely to be unaffected by the filter, whereas flickers with durations of 10 ms or less are greatly attenuated. Single-channel P o was calculated by dividing the summed open time with the total time (∼4 min). For dwell time analysis of ATP-gated CFTR, we used the previously described method . The probability density function (pdf) of the closed time distribution was generated from pooled closed events. This pdf consists of a sum of two exponential components. From the theoretical model and all the kinetic parameters (see Table I ), the areas and time constants of these two components were found by using a routine written by Dr. Gillis using MATLAB software (The MathWorks) following standard “Q-matrix” techniques . Curve fitting of the τc vs. ATP and single-channel P o vs. ATP relationships was performed using the Igor software and macros written to quantitatively describe these relationships (see discussion ). CFTR gating was studied in excised inside-out patches from NIH3T3 cells stably transfected with CFTR cDNA. In agreement with previous reports , the addition of ATP alone to the excised inside-out patches from NIH3T3-CFTR cells resulted in little channel activity, suggesting negligible basal phosphorylation of CFTR before patch excision. Application of the catalytic subunit of PKA (50 U/ml) with ATP (2.75 mM) activated a macroscopic current of ∼2.2 pA in this patch . Although this macroscopic current is the result of the activation of multiple channels, it is not so large as to be unable to resolve some of the single-channel steps. Since the single-channel amplitude is ∼0.4 pA and the P o under this condition is ∼0.4 (see below), it is estimated that 14 CFTR channels are present in this patch. Upon removal of both PKA and ATP, CFTR channel current rapidly decays in a monotonic manner. Readdition of ATP in the absence of PKA elicited a current magnitude approximately the same as that with ATP plus PKA, suggesting little dephosphorylation over that time span. Although we occasionally observed “rundown” of CFTR channels over long recording, presumably due to dephosphorylation by membrane-associated phosphatases, this is seldom a major problem in our attempt to quantify CFTR gating since patches showing significant rundown are discarded. Note an absence of any channel opening events for ∼15 s when ATP was completely removed. With the presence of 14 channels in the patch, this result suggests a negligible opening rate (<0.004 s −1 ) in the absence of ATP. It is known that the degree of CFTR phosphorylation can affect ATP-dependent gating , and that the presence of membrane-associated protein phosphatases can hamper accurate assessment of CFTR gating . We are fortunate that significant dephosphorylation of CFTR in the membrane patches does not occur in most of our experiments. Thus, when a steady state CFTR channel activity is reached upon addition of PKA and ATP into excised inside-out patches, CFTR channels should be highly phosphorylated and remain phosphorylated even when PKA is removed from the bath. This provides an opportunity to examine the ATP-dependent gating of strongly phosphorylated CFTR. Fig. 1 B shows a semicontinuous recording of a single CFTR in the presence or absence of PKA. The P o of the channel in the presence of PKA and ATP (2.75 mM) was 0.46. 2 or 10 min after removal of PKA, the P o remained unchanged (0.48 and 0.45, respectively). A second application of PKA at 19.5 min after PKA was removed did not alter the P o significantly. Thus, the CFTR channel behavior we observed likely reflects the gating pattern of strongly phosphorylated CFTR. To study ATP-dependent gating, we monitored the activity of phosphorylated CFTR channels in the presence of different concentrations of ATP. CFTR chloride channels in excised inside-out patches were first phosphorylated with PKA and ATP. To obtain an accurate dose–response relationship, we bracketed the experiments at tested concentrations of ATP with the responses of 2.75 mM ATP. Any minor rundown (<20%) was compensated for by comparing the channel activity in the presence of the tested concentration to the average activity in the presence of 2.75 mM ATP immediately before and after each tested concentration. The results of an experiment were disregarded when any rundown >20% was detected during a bracketed period. To ensure that we are observing the activity of fully phosphorylated CFTR throughout our experiments, we periodically compared the channel activity in the presence of 2.75 mM ATP to the activity in the presence of 2.75 mM ATP plus PKA . We assumed little or no dephosphorylation of the protein if no further increase in channel activity was detected upon the readdition of PKA. Fig. 2 A shows a representative trace with multiple phosphorylated CFTR channels exposed to two different concentrations of ATP. The mean current amplitude in the presence of 0.5 mM ATP is ∼80% that in the presence of 2.75 mM ATP. Fig. 2 B demonstrates the dose response of ATP-dependent gating by normalizing the macroscopic current at different concentrations of ATP to the current level at 2.75 mM. Since no alteration in single-channel amplitude is detected at different ATP concentrations (data not shown), it is concluded that the changes in the macroscopic current amplitude is due to alterations in the P o of individual channels. To demonstrate the effects of the ATP concentration on the single-channel P o , similar experiments were performed in patches containing a single CFTR channel. In these experiments, analysis was performed on recordings of strongly phosphorylated channels at least 4 min in length for each ATP concentration tested. Similar to the macroscopic current amplitude, ATP increased the single-channel P o in a concentration-dependent manner . The plot of single-channel P o vs. ATP concentration is very similar to the macroscopic dose–response relationship , suggesting that gating of CFTR is independent of the number of channels in the patches and that the macroscopic dose response is exclusively due to changes in gating at different ATP concentrations. To understand how changes in the ATP concentration affect CFTR gating, single-channel kinetics were examined. Dwell time analysis of CFTR channel activity in patches containing a single CFTR channel was performed. Fig. 3 A shows representative single-channel traces at four different [ATP]. It should be noted that openings that last for hundreds of milliseconds are interrupted by closings that vary with [ATP]. Even at 10 μM ATP, when there are only 10 openings in the 64-s trace shown, the openings are well dispersed and do not appear to occur in clusters. In addition, as the concentration of ATP is increased, some longer openings, although rare, can be seen. Fig. 3 B shows cumulative dwell time histograms from the data shown in Fig. 3 A. The closed time constant (τc) and open time constant (τo) were estimated from single exponential fit of the cumulative closed and open time histograms at each [ATP]. The relationship between τc and [ATP] follows a saturating function with no significant change in τc at ATP concentrations higher than 1.0 mM . This result suggests that at low micromolar concentrations of ATP, channels are not ATP-bound all the time; therefore, binding of ATP imposes a rate-limiting step in channel opening. However, once the binding of ATP to CFTR is saturated at ∼1 mM, the rate-limiting step in the opening of CFTR channels is some concentration-independent, post-binding event(s). To examine the closed time distribution in detail, we pooled all closed events in four patches in the presence of 100 or 500 μM ATP and generated the pdf of the closed time distribution . The closed time pdf resembles a single exponential distribution with the important exception that the maximal number of events is not at the minimal binned interval. The paucity of short-lived closings is particularly apparent at 100 μM ATP and is indicative of the presence of irreversible steps in ATP-dependent gating of CFTR . Fig. 4 B demonstrates that τo increases with increasing [ATP] . This ATP-dependent change in τo is inconsistent with the model proposed by Gunderson and Kopito . In the model of Gunderson and Kopito , the lifetime of the open state is determined by the hydrolysis rate of bound ATP at NBD2; therefore, τo is not predicted to change with [ATP]. In contrast, a prolongation of τo with increasing [ATP] is consistent with the model of Hwang et al. because it includes two open states with ATP binding at NBD2 leading to occupancy of the long-lived open state (see discussion ). Since this latter model predicts a prolongation of open times with increasing [ATP] due to ATP binding to NBD2, we next examined [ATP]-dependent gating of K1250A-CFTR, a mutant in which the conserved lysine in the Walker A motif of NBD2 is converted to alanine. This mutant CFTR has been shown to assume prolonged openings at 1 mM ATP presumably due to diminished hydrolysis of ATP at NBD2 . Gating of K1250A-CFTR channels was examined in the presence of either 10 μM or 2.75 mM ATP. Fig. 6 A shows that a K1250A-CFTR channel, preactivated with PKA and ATP (not shown), was “locked” in an open state with 2.75 mM ATP and the channel closed ∼2 min after ATP washout. In the same patch, very brief openings were seen when 10 μM ATP was applied. These short openings are the first evidence that the channel can close in the absence of ATP hydrolysis by NBD2 (see below). The single channel amplitude, obtained from the all-point histograms (not shown), of K1250A-CFTR channels opened with millimolar ATP is about the same as that for brief openings in the presence of 10 μM ATP . Note the presence of short flickering closures even in the absence of ATP , indicating that these closings are not coupled to an ATP hydrolysis cycle. Therefore, to quantify ATP-dependent gating events, these flickering closings should be excluded (see materials and methods ). To quantify the brief openings of K1250A-CFTR in the presence of 10 μM ATP, dwell time analysis of the cumulative open time from three different patches was performed. The cumulative histogram of pooled open times in the presence of 10 μM ATP could be fitted with a single-exponential function yielding a τo of 0.25 ± 0.01 s , which is close to the mean open time of wt-CFTR in the presence of 10 μM ATP. Since the “locked open” time of K1250A-CFTR is apparently very long , it will be very difficult to collect enough events for dwell time analysis. Even if we obtain patches containing a single K1250A-CFTR channel, the flickering closures in locked open state may interfere with the analysis. Although excluding closing intervals <80 ms is appropriate for eliminating flickers in analysis of wt-CFTR, this exclusion is not sufficient to eliminate flickers from analysis of K1250A-CFTR because the ratio of flickers to “true” closings (gating) of CFTR is much higher. We therefore estimate the time constant for the locked open state from the macroscopic current decay upon washout of 2.75 mM ATP. At this concentration, most of K1250A-CFTR channels are locked open, as can be judged from the small magnitude of macroscopic current fluctuations . The mean open lifetime of ATP-locked channels should be the same as the time constant for the current decay upon washout of ATP since the opening rate in the absence of ATP is negligible. To obtain a better estimation of the decay time constant, we repeated the ATP washout several times in the same patch and fitted the decay phase of ensemble macroscopic current with a single exponential function . From three different patches, the exponential decay of the macroscopic current upon washout of ATP yields a time constant of 162.6 ± 31.8 s (our solution change has a dead time of ∼3 s; see materials and methods ). These results suggest that, at low micromolar [ATP], K1250A-CFTR can assume brief openings with a time constant close to that of wt-CFTR at equivalent [ATP], but this mutant CFTR can be locked open for minutes at millimolar ATP. Assuming ATP is not hydrolyzed by the NBD2 of K1250A-CFTR, the slow closing rate reflects a slow dissociation of ATP from CFTR (presumably from NBD2). If the brief openings of K1250A-CFTR at 10 μM ATP represent open channel conformations without NBD2 being occupied, this observation suggests that CFTR can close even when ATP acts exclusively on NBD1 . To examine the rate of nucleotide unbinding from NBD2 of wt-CFTR, we used the nonhydrolyzable ATP analogue, AMP-PNP, which “locks open” CFTR presumably because it cannot be hydrolyzed by NBD2 to allow channel closure. To estimate the off rate of AMP-PNP from NBD2, we examined the time course of channel closure after washing out ATP/AMP-PNP mixtures that produce the locked open state. In Fig. 7 , 0.5 mM ATP was applied to elicit macroscopic PKA-phosphorylated CFTR channel current with a mean current amplitude of 1.16 pA in this patch. Addition of 0.5 mM AMP-PNP in the continued presence of ATP caused a 25% increase of steady state current. Once a new level of activity was obtained, both ATP and AMP-PNP were washed out, resulting in a biphasic decay of channel activity . At this one-to-one ratio of ATP and AMP-PNP, most of the channels are not locked open, as evidenced by the magnitude of current fluctuations at the steady state. In fact, from the slow closing steps after washout of nucleotides, it is estimated that only 1 or 2 channels had been locked open by AMP-PNP. In the same patch, however, 2.75 mM AMP-PNP caused a threefold increase in the macroscopic current elicited with 0.5 mM ATP. In spite of an approximately sixfold higher concentration of AMP-PNP, a significant number of channels are not locked open because large current fluctuations can be seen. Compared with the current decay phase upon removal of 0.5 mM AMP-PNP, however, more slow channel closing steps (representing closings from the locked open state) were observed. Although our solution change does not allow us to assess the fast phase of current decay, the slow phase of the current decay, which lasts for tens of seconds, can be quantified. To generate a macroscopic current decay, we again used ensemble CFTR current from the same patch. The slow phase of the current decay upon removal of 0.5 or 2.75 mM AMP-PNP was fitted with a single exponential function yielding a similar time constant of ∼30 s. This concentration-independent decay rate is consistent with the idea that the rate-limiting step for the current decay is the dissociation of AMP-PNP from NBD2, a concentration-independent process. These results also support the hypothesis that ATP binding to NBD2 stabilizes the open channel conformation. This stabilizing effect is greatly magnified when hydrolysis at NBD2 is eliminated, either through chemical modification of the binding molecule (AMP-PNP) or molecular alteration of the CFTR protein itself . Several linear equilibrium models have been proposed to explain ATP-dependent gating of CFTR . These models, all similar to models used to explain the kinetics of ligand-gated channels, do not consider the fact that CFTR is an ATPase and the likelihood that ATP hydrolysis provides the free energy that drives gating transitions. Since the free energy generated from hydrolysis of a single ATP molecule is >10 kT, it seems reasonable to model some of the ATP hydrolysis-driven conformational changes as essentially irreversible. Before we focus our discussion on asymmetrical, cyclic schemes that have been proposed to describe CFTR gating, we will first examine the feasibility of a simple equilibrium kinetic scheme by using our single-channel data. Assuming that ATP binding, but not hydrolysis, at NBD1 opens the channel, a simple scheme for a typical ligand-gated channel can be derived . According to this scheme, the transitions between C·ATP and O·ATP reflect the short flickers that we exclude from our analysis by omitting closings <80 ms. Thus, the only transitions we measure are between states C and C·ATP, and Fig. 8 , Scheme 1, predicts that our measured mean closed time is inversely proportional to [ATP] and therefore should approach 0 for high [ATP]. This is inconsistent with our observation of a minimum closed time of ∼400 ms at high [ATP] . In addition, Scheme 1 obeys microscopic reversibility and thus predicts that the pdf of the closed time distribution has two decay time constants. Our results with a rising phase in the pdf of the closed time distribution is inconsistent with Scheme 1 being an adequate model to explain CFTR channel gating. Three cyclic gating models for CFTR gating have been proposed whereby ATP hydrolysis at NBD1 is a prerequisite for channel opening . These models differ in their proposed roles for the two NBDs' participation in gating of the channel. Hwang et al. and Baukrowitz et al. proposed that for every ATP molecule hydrolyzed at NBD1, one opening event occurs (a strict coupling); subsequent binding at NBD2 stabilizes the open conformation; ATP hydrolysis at NBD2 leads to channel closure. Their model also suggests that channels can close even without a functional interaction between ATP and NBD2, although data supporting this notion are lacking. In contrast, Gunderson and Kopito propose that nucleotide binding and hydrolysis at NBD1 is only a prerequisite for channel opening; subsequent nucleotide binding at NBD2 is actually responsible for channel opening, and hydrolysis at NBD2 causes channel closure. The model proposed by Carson et al. is very similar to that by Hwang et al. except that hydrolysis at NBD2 is obligatory for channel closing, a feature shared by the model proposed by Gunderson and Kopito . All three models are supported by the observations that the nonhydrolyzable ATP analogues, such as AMP-PNP and ATPγS, fail to open CFTR channels, but dramatically increase the open time in the presence of ATP . The fact that mutations of the CFTR protein that impair nucleotide hydrolysis at NBD1 or NBD2 result in prolongation of channel closed or open times also supports these models. A critical observation in our current work for differentiating these models is the ATP concentration dependence of the mean open time . The model proposed by Gunderson and Kopito predicts that the mean open time is independent of [ATP] because the termination of an opening event is controlled by hydrolysis of the bound ATP at NBD2, which is an [ATP]-independent parameter. However, the model proposed by Hwang et al. predicts a second prolonged open state when ATP binds to NBD2. Thus, two open time constants reflecting the life times of these two open states are expected. We are unable to clearly separate two exponential components in the open dwell time histograms for wt-CFTR . Nevertheless, we observe a shift of the mean open time as [ATP] is increased. Our failure to detect a double exponential distribution of open times does not necessarily eliminate the possibility of two open states. If the difference in the mean lifetime of two open states is small and/or the transition between two open states is slow, then technically it will be difficult to collect sufficient events to resolve these two time constants. Furthermore, our exclusion of “flickers” with the 80 ms minimal duration criterion may also distort the resolution of the two components. We clearly resolve two open times with K1250A-CFTR and demonstrate a dramatic [ATP] dependence of the channel open time for this mutant CFTR. One interesting observation is that at 10 μM ATP, the mean open time for K1250A-CFTR is ∼250 ms, a value very close to that for wt-CFTR at the equivalent [ATP]. Thus, although this NBD2 mutant CFTR is able to open for minutes at high concentrations of ATP, it can assume very brief opening (∼250 ms) at low micromolar [ATP]. This observation is expected if the on rate of ATP binding to NBD1 is much larger than that for NBD2, and therefore at a low concentration of ATP, ATP interacts with NBD1 exclusively. Based on this interpretation, the brief opening with K1250A-CFTR is coupled to hydrolysis of one ATP molecule at NBD1 and subsequently the channel can close without ATP hydrolysis at NBD2. This conclusion is in conflict with the model for the obligatory role of ATP hydrolysis at NBD2 in channel closings . This same observation that the open time of K1250A-CFTR depends on [ATP] is also inconsistent with the proposal that ATP binding at NBD2 opens the channel . According to this latter model, every opening of K1250A-CFTR should last for minutes. From the above discussion, we conclude that the model proposed by Hwang et al. provides a reasonable background to construct a more quantitative scheme with the kinetic parameters obtained from the present work. First, the spontaneous opening of phosphorylated CFTR in the absence of ATP is negligible , suggesting that only ATP-bound channels (C·ATP in Scheme 2) can advance to the open state. Since the input of energy is involved in opening the channel, we propose that the transition from the ATP-bound closed state to the open state is a relatively irreversible process. Since NBD1 alone allows channel opening as well as closing (see above), we propose that the state O sojourns in C directly. This will be another irreversible step since the C to O transition (i.e., opening CFTR in the absence of ATP) is negligible. Fig. 8 , Scheme 2, summarizes the coupling of ATP hydrolysis at NBD1 to channel gating. At [ATP] ≥ 1 mM, the ATP binding step is saturated. A maximal opening rate of 1.92 s −1 , the reciprocal of the averaged closed time at millimolar [ATP] , should be equal to k 1 , the intrinsic opening rate for an ATP-bound channel. Although the open time constants at different [ATP] are the weighed average of the lifetimes for the two open states, O and O·ATP (see below), the mean open time at a very low [ATP] should represent a closing of the channel through ATP action at NBD1 only. We estimate this short open time constant by extrapolating the first five data points in Fig. 4 B to 0 mM ATP. The reciprocal of this short open time constant (0.26 s) should be equal to k 2 (3.85 s −1 ), the intrinsic closing rate of the open channel by NBD1. The fact that this short open time constant is very close to the mean open time of K1250A-CFTR at 10 μM ATP further supports this assignment. Assuming a steady state condition for Fig. 8 , Scheme 2, we obtained Eq. 1 to describe the relationship between [ATP] and τc . Fitting Eq. 1 to the data of Fig. 4 A yields values of 75,600 s −1 M −1 and 6.5 s −1 for α 1 and β 1 , respectively. It should be noted that, although changing α 1 or β 1 individually alters the best fit drastically, when the K d1 (β 1 /α 1 ) is kept constant (86 μM), the best fit does not change significantly with parallel changes in α 1 and β 1 over a 20-fold range (data not shown). Thus, the absolute values for α 1 and β 1 are less certain than the value of 86 μM for K d1 . However, α 1 is not likely smaller than 1,000 M −1 s −1 since the fitted curve starts to deviate from the actual data points at low [ATP] (not shown). Even though Eq. 1 is based on an assumption of steady state (which, strictly speaking, cannot be true for a single molecule), the values for α 1 and β 1 are likely appropriate for two reasons. First, the mean closed times at different [ATP] found from the exact solution of the Q matrix , were very close to the closed times calculated from Eq. 1 . Second, these values for k 1 , k 2 , α 1 , and β 1 also reasonably fit the pdf of the closed time distributions (including the rising phase and the position of the peak) at 0.1 and 0.5 mM ATP : 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\tau}_{c}=\frac{{\alpha}_{1}[ATP]+{\beta}_{1}+k_{1}}{{\alpha}_{1}[ATP]k_{1}}.\end{equation*}\end{document} To expand Fig. 8 , Scheme 2, to incorporate the function of NBD2 , we assume that ATP can only bind to the open conformation of CFTR . We also assume that ATP hydrolysis at NBD2, instead of closing the channel directly, enables the channel to close through the state O . This assumption, although somewhat arbitrary, simplifies the biochemical interpretation of the gating transitions (see below). As discussed above, the rate-limiting step for channel closing from the AMP-PNP locked open state is likely the dissociation rate of AMP-PNP. Since the structure of AMP-PNP is very similar to that of ATP, we assume that the off rate for ATP at NBD2 for wt-CFTR is the same as that for AMP-PNP. Our estimation of the off rate for AMP-PNP from the macroscopic current decay upon removal of nucleotides is 0.03 s −1 . A small β 2 value suggests that channel closings from O·ATP almost always sojourn in the state O via hydrolysis and dissociation of the hydrolytic products from NBD2. Since the off rate of AMP-PNP at NBD2 is very slow, AMP-PNP locked open state can be considered an absorbing state. A reasonably fast on rate of AMP-PNP should lead to most channels locked in that state. However, even at 2.75 mM AMP-PNP, not all channels were locked open , suggesting a very slow on rate of AMP-PNP (and presumably also ATP) to NBD2. The steady state P o for CFTR in the presence of 0.5 mM ATP is ∼0.28. A 3.18-fold increase of the macroscopic current by addition of 2.75 mM AMP-PNP indicates a P o of 0.89 in the presence of 0.5 mM ATP and 2.75 mM AMP-PNP . This P o is determined by the fraction of channels in the open state and the fraction of channels in the locked open state. Since the opening rate and closing rate of CFTR at 0.5 mM ATP, and the closing rate of AMP-PNP locked open channels are known , the association rate constant for AMP-PNP is estimated to be 109 s −1 M −1 . Assuming ATP has similar on and off rates as AMP-PNP to NBD2, an ATP dissociation constant, K d2 (β 2 /α 2 ), of 275 μM is obtained for NBD2. Finally, we used the equation (Eq. 2 ) that describes the relationship between the steady state P o and [ATP] to obtain the k 3 value . Fitting Eq. 2 with the data using all the kinetic parameters derived above yields a k 3 value of 1.78 s −1 . This value has some degree of uncertainty because P o is not very sensitive to NBD2 function in our excised patch experiments. Table I summarizes all the estimated values for the kinetic parameters and their experimental origins. It seems surprising that even at 10 mM ATP, the on rate of ATP to NBD2 is ∼1 s −1 , suggesting a minimal contribution of NBD2 to P o of CFTR in our experiments using excised inside-out patches. This puzzling lack of role of NBD2 may be related to some of the discrepancies among different reports discussed below. \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{o}=\frac{{\alpha}_{1}[ATP]{\beta}_{2}k_{1}+{\alpha}_{1}[ATP]k_{1}k_{3}+{\alpha}_{1}{\alpha}_{2}[ATP]\hspace{.167em}^{2}k_{1}}{{\alpha}_{1}[ATP]{\beta}_{2}k_{1}+{\alpha}_{1}[ATP]k_{1}k_{3}+{\alpha}_{1}{\alpha}_{2}[ATP]\hspace{.167em}^{2}k_{1}}\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{{\alpha}_{1}[ATP]{\beta}_{2}k_{1}+{\alpha}_{1}[ATP]k_{1}k_{3}+{\alpha}_{1}{\alpha}_{2}[ATP]^{2}k_{1}}{\;+{\alpha}_{1}[ATP]{\beta}_{2}k_{2}+{\alpha}_{1}[ATP]k_{2}k_{3}+{\beta}_{1}{\beta}_{2}k_{2}+{\beta}_{1}k_{2}k_{3}}\end{equation*}\end{document} 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{{\alpha}_{1}[ATP]{\beta}_{2}k_{1}+{\alpha}_{1}[ATP]k_{1}k_{3}+{\alpha}_{1}{\alpha}_{2}[ATP]^{2}k_{1}}{\;+{\beta}_{2}k_{1}k_{2}+k_{1}k_{2}k_{3}}\end{equation*}\end{document} A discrepancy exists between our maximal P o measured in excised inside-out patches and previous reports with higher P o values. When CFTR gating was examined in excised giant patches from cardiac myocytes, a P o ≥ 0.8 can be achieved in the presence of PKA and 0.5 mM ATP . This is not likely due to species or tissue differences because a similar high P o was observed with human wt-CFTR incorporated into lipid bilayers . In fact, the P o of wt-CFTR in cell- attached patches from Calu-3 cells, a human airway epithelial cell line, can be >0.9 in the presence of cAMP stimulation . This latter result is especially enigmatic. In cell-attached patches, in addition to the ATP-dependent gating events, phosphorylation/dephosphorylation events need to be considered in determining P o . Since only phosphorylated CFTR channels can be opened by ATP, a P o of ∼0.5 in inside-out patches for the strongly phosphorylated CFTR should impose a maximal P o of ∼0.5 for CFTR activity measured in cell-attached patches where at least one more closed state (dephosphorylated closed state) should be considered in calculating P o . Furthermore, the channel open times in cell-attached patches can be easily resolved into two components , and the longer time constant is greater than the mean open time at 10 mM ATP in excised patches from the current study. One interesting possibility is that a cytosolic factor, which affects CFTR gating, is missing in our excised patches but remains in other systems . Numerous reports on CFTR gating have generated many different numerical values for the kinetic parameters. This difference likely reflects the difference in methods used for kinetic analysis. Importantly, different cutoffs are used to define CFTR gating events, resulting in a wide range of the time constants (2 ms–3 s). Interestingly, the steady state P o values reported for wt-CFTR in those reports were not very different (0.28– 0.65). Very few studies have assigned those time constants to specific kinetic events, perhaps because there often is an inconsistency between kinetic parameters, determined by dwell time analysis, and the independently measured steady state P o . For example, Li et al. , using 60 ms as a cutoff, reported a P o of 0.48 at 1 mM ATP for purified wt-CFTR incorporated into lipid bilayers, whereas the kinetic analysis shows a mean burst duration (τb) and a mean interburst duration (τib) of 600 and 125 ms, respectively, which would predict a P o of 600/(600 + 125), or 0.83. An earlier report from the same group showed a P o of 0.28 with a τb of 0.36 s and τib of 3.35 s . Clearly, these values of the kinetic parameters cannot easily be reconciled with the P o value. Carson et al. , using 20 ms as a cutoff, reported a mean burst time of ∼1 s for K1250A-CFTR. This number is evidently an underestimation of the true ATP-coupled open time for K1250A-CFTR as a continuous burst of opening that lasts for minutes is observed even when ATP is removed . On the other hand, we believe that the method we used for macroscopic as well as microscopic kinetic analysis provides a reasonable approximation of the [ATP]-dependent changes in P o . Other than the amino acid sequences in the conserved Walker A and B motifs, NBD1 and NBD2 share only ∼30% sequence homology . Our results, as well as others, suggest that, functionally, they play very different roles in CFTR gating. These distinct functional roles for CFTRs NBD1 and NBD2 also distinguish CFTR from p-glycoprotein, another member of the ATP-binding cassette (ABC) transporter family. The two NBDs in p-glycoprotein share 66% sequence homology and there is no evidence so far to support that NBDs in p-glycoprotein are functionally distinct. Biochemical studies suggest that two NBDs of p-glycoprotein may alternate in catalyzing ATP hydrolysis reactions . This is not very surprising for members of the ABC family other than CFTR since structural (perhaps functional) symmetry is recently proposed from the x-ray crystallographic structure of His permease . Thus, although CFTR shares topological similarities with other proteins in this family, numerous differences may have emerged during evolution. Our data also suggest differences in ATP binding affinity between NBD1 and NBD2. For NBD1, ATP has a much faster on as well as off rate. However, the association rate constant for NBD2 is slower than diffusion by several orders of magnitude, suggesting the rate constant, α 2 , obtained from our analysis does not truly represent the association rate constant for a simple bimolecular reaction. One possible explanation for this apparent slow on rate of ATP to NBD2 can be inferred from the analogy with G proteins. If the function of NBD2 indeed parallels that of a G protein, ADP, the hydrolytic product of ATP hydrolysis, may be entrapped after hydrolysis. In analogy to the G protein function, occupancy of this ADP molecule in NBD2 may prevent ATP binding. If this analogy is applied to our model , then the state O contains an ADP molecule in NBD2. A similar proposal was recently postulated by Senior and Gadsby . Thus, the apparent slow on rate for ATP binding to NBD2 is caused by the slow dissociation of ADP, and this slow off rate sets an upper limit for ATP association with NBD2. As discussed above, our kinetic data fail to explain some of the previous observations that the P o of CFTR can reach 0.8–0.9 . We do not know the mechanism that can account for these discrepancies. Perhaps the hypothetical factor that we lose upon patch excision accelerates the ATP/ADP exchange rate. Thus, much of the role of NBD2 in determining P o is lost in excised inside-out membrane patches. Again, in analogy to G proteins, for example, GDP binds very tightly to the G protein Ras, but the GTP/ GDP exchange rate is greatly increased by the presence of the Sos protein . Two irreversible steps in gating transitions are proposed to be coupled to ATP hydrolysis at NBD1 . A paucity of short closings in the pdf of the closed time is predicted with a scheme such as Scheme 2 that violates microscopic reversibility. Baukrowitz et al. proposed that CFTR channels open after ATP is hydrolyzed. According to this proposition, we speculate that the splitting of the γ-phosphate from ATP yields one irreversible step with the rate constant, k 1 , and the release of the hydrolytic products, phosphate or ADP or both, can generate a second irreversible step with the rate constant, k 2 . Then the intrinsic hydrolysis rate for NBD1 is k 1 k 2 /( k 1 + k 2 ) = 1.28 s −1 . This value is comparable to the reported hydrolysis rate of ∼1 s −1 per CFTR molecule . Our model also suggests that the ATP hydrolysis reactions in NBD1 and NBD2 are coupled. Hydrolysis of ATP at NBD2 requires occurrence of an earlier ATP hydrolysis reaction at NBD1; while part of the ATP hydrolysis cycle at NBD1 (the O → C transition) can be disrupted by ATP occupancy at NBD2 . One prediction from this model is that abolition of the ATP hydrolysis rate at NBD2 will affect the overall ATP hydrolysis by CFTR. Assuming the opening rate of K1250A-CFTR is the same as that of wt-CFTR, our kinetic data suggest a maximal ATP hydrolysis rate of ∼0.005 s −1 , which is 1/200 of that for wt-CFTR. Recent preliminary data that K1250A-CFTR has a drastically reduced rate of ATP hydrolysis support this coupled ATP turnover hypothesis . One should note, however, that our model, although sufficient to explain numerous biophysical and biochemical results, simply provides a testable framework for future exploration to advance our understanding of the biochemical basis of CFTR gating via ATP hydrolysis. | Study | biomedical | en | 0.999998 |
10102936 | Strong inward rectifier K channels such as Kir2.1 (IRK1) play a key role in setting the resting membrane potential and regulating excitability in heart, neurons, and many other cell types . The strong inward rectification property has been previously attributed to intracellular Mg and polyamines directly plugging the channel pore at membrane potentials positive to the K equilibrium potential, causing voltage-dependent open channel block . Detailed analysis of block by spermine, however, requires a fairly complex model to account for all the voltage dependence and kinetic features . Moreover, open channel block as the sole mechanism of inward rectification has been questioned in a recent study in which Kir2.1 channels were reconstituted into lipid bilayers . In the absence of Mg or polyamines, the channels still exhibited rectification. Mg or polyamines enhanced inward rectification, but the mechanism of block was more complex than could be modeled by simple open channel block. We have previously described an intrinsic pH-sensitive gating mechanism of inward rectification in Kir2.1 in the absence of Mg or polyamines . This was manifested as a persistent voltage- dependent inactivation of outward currents in excised patches after prolonged washout in polyamine- and Mg-free solution, which was accelerated by raising intracellular pH to 9.0. The intrinsic gating mechanism is kinetically much slower than polyamine or Mg block and is most obvious only with large depolarizations (to more than +40 mV), explaining why it was only subtly apparent in some previous studies and assumed to represent residual polyamine block . Spider venom polyamine toxins, philanthotoxin and argiotoxin, have been shown to block N -methyl- d -aspartate receptors extracellularly . Since polyamines cause inward rectification in Kir channels, we investigated the effects of these toxins on the strong inward rectifier channel Kir2.1 (IRK1) to obtain further mechanistic insight into polyamine-induced inward rectification. The toxins are structurally similar to spermine at one end and have a bulky hydrophobic aromatic group at the other . By studying competitive interactions between polyamines and these polyamine toxins, we present evidence that polyamine block is more complex than direct open channel block. Our results suggest that direct open channel block accounts for low affinity block by spermine and spermidine, but high affinity block by these polyamines involves their binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. In vitro T7-transcribed cRNA (Ambion Inc.) was injected (50– 5,000 ng) into stage V–VI Xenopus oocytes isolated by partial ovariectomy under tricaine anaesthesia. PCR mutagenesis was carried out by the overlap extension technique . Patch clamp studies were performed at room temperature on giant excised inside-out patches, as described previously . The patch electrode contained (mM): 100 KOH, 1.8 CaCl 2 , and 5 HEPES, pH 7.4 with 100 MES. The standard bath solution contained (mM): 100–104 KCl + KOH, 5 EDTA, and 5 HEPES, adjusted to pH 7.2 with MES. Polyamines and polyamine toxins were added directly to the bath solution. Rapid bath solution changes (<1 s) were accomplished using a rapid solution exchange device . All current traces shown in the figures were leak corrected by subtracting currents recorded with 30 mM tetraethylammonium in the bath solution, which blocked Kir2.1 currents completely . Curve-fitting and model simulations were performed using Sigmaplot version 2.0 (Jandel Scientific) and SCoP version 3.5 (Simulation Resources, Inc.). Kir2.1 channels were heterologously expressed in Xenopus oocytes. When applied to the cytoplasmic surface of giant inside-out patches, both philanthotoxin and argiotoxin blocked outward currents through Kir2.1 channels in a voltage-dependent manner. At +40 mV, the concentrations of philanthotoxin and argiotoxin producing half-maximal block ( K 0.5 ) were 47 nM ( n = 4 patches), and 37 nM ( n = 4), respectively . Unlike N -methyl- d -aspartate receptors, spermine blocked Kir2.1 channels with higher affinity ( K 0.5 of 5.9 nM at +40 mV) than either toxin . Since earlier mutagenesis studies have defined two key negatively charged amino acids, D172 in the M2 region and E224 in the COOH terminus, as regulators of polyamine- and Mg-induced inward rectification in Kir2.1 , we also tested the effects of philanthotoxin on the mutants D172N and E224G. Both mutants were considerably less sensitive to philanthotoxin block than wild-type Kir2.1, with K 0.5 values of 1,220 nM for D172N and 7,090 nM for E224G . These findings are generally consistent with the long-pore plugging model of polyamine block , in which polyamines such as spermine are proposed to insert lengthwise into the channel pore. The polyamine end of the toxin could also insert lengthwise, but would be unable to insert crosswise because of the large hydrophobic end. We next investigated interactions between spermine and philanthotoxin block. A fixed concentration of philanthotoxin (30 nM) was chosen that partially blocked steady state outward current at +40 mV, by 44% . In the presence of 30 nM philanthotoxin, the concentration of spermine required to block the remaining current by 50% ( K 0.5 ) increased from 5.9 to 78 nM . The same effect was seen with the trivalent polyamine spermidine , whose K 0.5 increased from 31 nM in the absence of philanthoxin to 342 nM ( n = 5 patches) when 30 nM philanthotoxin was present. Finally, argiotoxin (10 nM) also interfered with spermine block of outward Kir2.1 currents to a comparable extent as philanthotoxin, increasing the K 0.5 for spermine from 5.1 to 53 nM ( n = 5 patches) at +40 mV . The ability of polyamine toxins to desensitize Kir2.1 channels to block by spermine or spermidine to this degree is difficult to reconcile with a direct open channel block mechanism in which toxin and polyamine molecules compete independently for binding site(s) in the pore. In the long-pore plugging model , it is hypothesized that two spermine molecules directly occupy the pore, producing either shallow (B1) or deeply bound (B2 and B3) states. We incorporated two additional blocked states to represent direct block by a single toxin molecule occupying the pore (the B4 state), and a toxin molecule capping a deeply bound spermine molecule (the B5 state). 10 rate constants (five equilibrium constants) connect the various states. At a concentration of toxin producing 44% block, this model does not produce a large shift in the K 0.5 for spermine . A small increase in the K 0.5 for spermine (1.8-fold) can be produced if the B5 state is disabled . As the affinity of the toxin for the B5 state is increased, however, the K 0.5 for spermine progressively shifts to lower rather than higher values. The same predictions were obtained with simpler models of direct open channel block, with totals of two to four blocked states. These results indicate that block of Kir2.1 channels by polyamines and polyamine toxins is more complex than a direct open channel block mechanism . An intrinsic gating mechanism has previously been proposed to explain inward rectification of K channels . Recently we and others have characterized an intrinsic gating mechanism in the cloned Kir2.1 channels in which persistent inactivation of outward current occurs in the absence of polyamines or Mg. Fig. 4 shows the time course of inactivation of outward Kir2.1 currents in giant inside-out patches excised into a polyamine- and Mg-free solution. Over the first 5 min, there was a time-dependent decrease in the rate of inactivation of outward current (measured at +60 mV), previously attributed to the washout of endogenous polyamines , but thereafter no further change occurred. During the polyamine- and Mg-free washout period, if Kir2.1 currents were first inactivated by depolarizing the membrane to +60 mV, and then rapidly unblocked by hyperpolarizing to −30 mV, a subsequent depolarization to +60 mV still produced inactivation of outward current. This seems inconsistent with a blocking molecule lodged in the pore of the channel, since it should diffuse away once released from its blocking site during hyperpolarization, and not be available to reblock the channel upon the next depolarization (unless a compartmentalized pool of poorly diffusible polyamines is postulated). An alternative explanation is that polyamines bind to and enhance the effectiveness of an intrinsic gate. The polyamine molecule could remain tethered to the intrinsic gate when released from the pore by hyperpolarization, and thereby be available to reocclude the pore during the subsequent depolarization. To determine whether this type of mechanism could account for the philanthotoxin-spermine interaction, we examined the model schematically illustrated in Figs. 4 C and 5 A. We hypothesize that, due to its linear structure with four symmetrically spaced positive charges , spermine binds at one end to the intrinsic gate, and the other end to a docking site in the ion-conducting pathway . This mechanism is assumed to account for the high affinity voltage-dependent block by spermine, whereas direct interaction of free, untethered spermine with the pore docking site produces lower affinity block. Philanthotoxin, however, can only block Kir2.1 channels when its spermine-like end lodges in the docking site in the pore . If the spermine-like end of philanthotoxin binds to the intrinsic gate, the hydrophobic end is too bulky to reach and interact with the docking site in the pore. This hypothetical schema predicts an interaction between polyamine toxins and spermine: if, in the presence of a polyamine toxin, the putative site in the intrinsic gate to which spermine binds is occupied by the spermine-like end of the polyamine toxin molecule, then spermine will no longer have access to its high affinity blocking mechanism. Thus, spermine's ability to block Kir2.1 channels with high affinity should be reduced. The simplest model of interaction between a polyamine toxin (T), spermine (S), and an intrinsic gate is shown in Fig. 5 A. Three open states reflect either nothing (O1), spermine (O2), or a toxin molecule (O3) bound to the intrinsic gate site, with the pore docking site empty. There are seven possible blocked states: the B1 state represents high affinity block by the spermine-intrinsic gate complex occluding the pore. The B2, B3, and B4 states represent an untethered spermine molecule bound to the docking site in the pore (direct low affinity block) with either nothing, spermine, or a toxin molecule, respectively, bound to the intrinsic gate. The B5, B6, and B7 states represent the analogous conditions for a toxin molecule directly blocking the pore. As in the long-pore plugging model , 10 rate constants connect the various states, represented in the diagram by their corresponding equilibrium constants K 1 – K 5 . Based on the difference in affinities for spermine block between wild-type Kir2.1 and the E224G mutation , we chose K 0.5 for high affinity block by the spermine-intrinsic gate complex to be ∼1,000× higher than direct block by untethered spermine molecules. By altering the affinity of the toxin for the intrinsic gate (i.e., adjusting the K 3 equilibrium constant), the experimentally observed decreased affinity of spermine block in the presence of toxin is readily reproduced by the model, and yields an acceptable fit to the data . The increase in K 0.5 in the presence of toxin is explained by the O3 state being favored as the affinity of the toxin for the intrinsic gate is increased, shifting the equilibrium away from spermine's high affinity blocking pathway (O1 → O2 → B1). In contrast, a direct open channel block mechanism, in which both toxin and spermine compete independently for a binding site in the pore, provides no means for the toxin to shift the equilibrium towards an open state, and so cannot account for the observed shift in the K 0.5 for spermine in the presence of philanthotoxin. Although we have not tested for toxin–toxin interactions, we would predict minimal interaction between different polyamine toxins, since neither would have access to the high affinity blocking route coupled to the intrinsic gate. The philanthotoxin–spermine competition experiments strongly support the role of an intrinsic gate in high affinity polyamine block of Kir2.1 channels. Since D172 and E224 have both been shown to be important in regulating the sensitivity of Kir2.1 to block by polyamines and Mg , we also examined the philanthotoxin–spermine interaction in the D172N and E224G mutants. For D172N, the K 0.5 for spermine at +40 mV increased from 5.9 to 90 nM ( n = 4), and for philanthotoxin, from 47 to 1,220 nM ( n = 6), compared with wild-type Kir2.1 . In the presence of 700 nM philanthotoxin, which blocked outward current at +40 mV by 40%, the K 0.5 for spermine increased from 90 to 1,050 nM . A possible interpretation of these findings is that D172 regulates binding of polyamines and toxin to the pore docking site . By collectively destabilizing the binding to this site of the spermine-intrinsic gate complex, untethered spermine and philanthotoxin, D172N should exhibit lower affinity block by both spermine and philanthotoxin. Assuming that the highest affinity block still occurs via the spermine-intrinsic gate complex, however, the ability of philanthotoxin to decrease the affinity of spermine block should still be preserved. All of these findings were obtained in the D172N mutant. By appropriately adjusting the equilibrium constants to reflect the lower affinities at the pore docking site, these effects were readily simulated in the intrinsic gating model . In contrast, the direct block model again failed to reproduce the philanthotoxin–spermine interaction (not shown). For E224G, the K 0.5 s for block by spermine and philanthotoxin were also both increased, from 5.9 to 6,030 nM ( n = 4) and from 47 to 7,090 nM ( n = 5), respectively . However, in the presence of 3,000 nM philanthotoxin, which blocked outward current at +40 mV by 31%, the increase in the K 0.5 of spermine block, as seen in both wild-type Kir2.1 and the D172N mutant, was eliminated (6,030 nM in the absence of philanthotoxin versus 4,800 nM in its presence, n = 5). A possible interpretation of these findings is that E224 primarily regulates polyamine binding to the intrinsic gate. If the E224G mutation impairs binding of spermine and philanthotoxin to the intrinsic gate, then block can occur only through direct interaction of untethered spermine or philanthotoxin with the pore docking site (near D172). The state diagram for the E224G mutant then reduces to a direct open channel block model containing only one open state and two blocked states (B2 and B5); i.e., a simplified version of Fig. 3 A. In this case, the spermine–philanthotoxin interaction should be markedly reduced. Exclusive assignment of E224's role to the polyamine binding site on the intrinsic gate, however, does not account for the increase in the K 0.5 for philanthotoxin block in the E224G mutant. Thus, E224 must also contribute to the stability of philanthotoxin and, perhaps, spermine binding to the pore region. This is consistent with previous observations showing that the E224G mutation affects single channel properties through the Kir2.1 pore . These findings support the intrinsic gate model of spermine block hypothesized in Figs. 4 C and 5 A, in which the D172 residue regulates the docking site in the pore, and E224 regulates the polyamine binding site on the intrinsic gate in addition to influencing binding of polyamine toxins (and probably also polyamines) in the pore. The evidence can be summarized as follows: (a) an intrinsic gating mechanism of inward rectification in Kir2.1 has been previously identified , which persists indefinitely after washout of endogenous internal polyamines or Mg . (b) Polyamines remain available to reblock Kir2.1 channels after they are released from their blocking site in the pore by hyperpolarization , suggesting that they remain tethered to another region of the channel from which they dissociate slowly. (c) Polyamine toxins, such as philanthotoxin and argiotoxin, interfere with the ability of spermine or spermidine to block Kir2.1 channels . This effect cannot be explained by a direct open channel block mechanism , but is readily accounted for by an intrinsic gate model . In the latter, high affinity block is due to a spermine-intrinsic gate complex, and low affinity block by direct open channel block of untethered spermine molecules. (d) The D172N mutation reduces the affinity of the channel for block by both spermine and philanthotoxin, but does not eliminate the spermine–philanthotoxin interaction, suggesting its major effect is destabilization of the pore docking site for all three blocking entities (the spermine-intrinsic gate complex, untethered spermine, and philanthotoxin). (e) The E224 mutation eliminates the spermine–philanthoxin interaction, consistent with destabilization of spermine's binding to the intrinsic gate. The assignment of D172 to the pore docking site, and E224 primarily to the intrinsic gate binding site may explain why two such widely separated amino acids play key roles in rectification. Several limitations of this study should be recognized. We cannot absolutely exclude the possibility of a compartmentalized pool of polyamines remaining after extensive washing of excised patches. However, such a pool would have to have multiple time constants with fast and very slow (>>15 min) components , and cannot account for the philanthotoxin–spermine interaction by a pure open channel block mechanism . In the model proposed in Fig. 5 A, the analysis was simplified by omitting the blocked state corresponding to the intrinsic gate unoccupied by a spermine molecule, even though outward current was significantly attenuated at the end of the voltage clamp pulse to +40 mV in the absence of polyamines or Mg . Long voltage clamp pulses (200 ms) were therefore used to allow this process to reach quasi-steady state before measuring the effects of polyamines and polyamine toxins on the remaining current. Our analysis was restricted to equilibrium conditions at +40 mV, and we have not yet evaluated whether the intrinsic gate model can account for the complex kinetics and voltage dependencies characterized extensively by Lopatin et al. . The substantial block by the intrinsic gating mechanism itself (in the absence of polyamines) complicates this analysis. Whether Mg or other polyamines, such as putrescine, bind to or use this putative intrinsic gate mechanism has also not yet been determined. Although we think it likely based on earlier analysis of Kir2.1 mutants , we have not proven that the intrinsic gate that weakly blocks outward current in the absence of polyamines is the same structure to which polyamines bind to cause high affinity block. The molecular configuration of the putative intrinsic gate region and even number of intrinsic gates per channel (given the homotetrameric structure of Kir2.1) remains purely speculative; it is solely for clarity and convenience that the intrinsic gate has been drawn as a single tethered gating particle in Figs. 4 C and 5 A. In fact, the dual effect of the E224G mutation on both the polyamine binding site on the intrinsic gate and its docking site in the pore is difficult to reconcile physically with such a simple model. At first glance, Fig. 5 A appears complicated. However, the polyamine block mechanism (shaded area) has the same number of rate constants and only one additional state compared with the direct block model Fig. 3 A. Incorporating the toxin–spermine interaction adds four additional states compared with the direct block model, but retains the same number of rate constants. Finally, whether the intrinsic gate mechanism also applies to other members of the strong inward rectifier K + channel family is currently unknown. In summary, these findings support the idea that a region of the Kir2.1 channel acts as an intrinsic gate that binds a polyamine molecule such as spermine or spermidine to induce strong inward rectification. Multiple positive charges on the polyamine molecule are likely to facilitate the voltage-dependent interaction of the intrinsic gate–polyamine complex with a pore docking site. Lower affinity block is produced by direct open channel block by untethered polyamines. The intrinsic gate mechanism retains the important feature of the strong inward rectifiers, relief of inward rectification by elevated K, hypothesized to result from destabilization of the polyamine-intrinsic gate docking site in the pore by external K ions. In view of recent structural information about pore dimensions of inward rectifier K channels , the intrinsic gate is an appealing modification to the direct block long pore plugging mechanism , since it requires only one spermine molecule, either tethered or untethered, to block the pore. With the distance from the internal surface to the selectivity filter estimated at 3 nm , a single spermine molecule occupying this section of the pore is reasonable, but more problematic for a direct block mechanism requiring two spermine molecules stacked lengthwise on top of one another (4 nm long) to fit into this region. | Study | biomedical | en | 0.999998 |
10102937 | Human ether-à-go-go –related gene ( HERG ) 1 encoded K + channel isoforms exist in a number of cell types including neurons, glia, cardiac myocytes, and tumor cells . HERG channels may be involved in neuronal spike frequency adaptation and tumor cell growth . In the heart, HERG K + channel subunits mediate a delayed rectifier K + current (I Kr ) that aids cellular repolarization . Mutations of the HERG gene or drugs that suppress cardiac I Kr cause the congenital and acquired forms of human long QT syndrome, respectively . Despite homology to the Shaker family of voltage gated K + channels, HERG K + channels exhibit distinctive gating behavior. Voltage-dependent channel activation is much slower than channel inactivation, giving rise to the characteristic rectification-like behavior of this channel . There is also evidence that HERG channel inactivation has intrinsic voltage dependence , unlike N- and C-type inactivation in the Shaker family K + channels . Divalent cations have often been used to probe ion channel function, and Ca 2+ has been implicated as a modulator of K + channel gating . Interaction between Ca 2+ and a distinct region of the channel protein could cause a specific effect on its function, or Ca 2+ could nonspecifically associate with diffuse charged moieties on the membrane surface, effectively neutralizing charges and leading to a global change in the transmembrane potential perceived by all membrane molecules. Both mechanisms of modulation have been proposed to explain the effects of divalent cations on ion channels . In the squid axon, elevation of extracellular Ca 2+ leads to a decrease in the rate of K + channel activation . Complete removal of extracellular divalent cations and K + causes loss of selectivity and gating in the squid axon K + channel, resulting in channels that are constitutively open. Recombinant K + channels from Drosophila melanogaster, including Shaker and EAG, are affected by divalent cations in much the same way as the squid K + channel . Unlike invertebrate K + channels, guinea pig ventricular I Kr channels appear to retain gating and selectivity in the absence of extracellular Ca 2+ and K + . However, Ca 2+ and Mg 2+ have been suggested to serve critical roles in the gating of K + channels from both rabbit sinoatrial node and neuronal cell lines, and these channels are functionally similar to HERG K + channels . To gain further insight into the gating behavior of HERG K + channels, we have examined the effect of external Ca 2+ on HERG channels under conditions in which the extracellular K + concentration was in the normal physiological range (4 mM). The HERG cDNA was obtained from Dr. Mark Keating (University of Utah, Salt Lake City, UT). HERG was ligated into the pSI mammalian expression plasmid ( Promega Corp. ). The CD8 antigen gene in the EBO-pcD Leu2 vector was kindly provided by Dr. Richard Horn (Thomas Jefferson Univ. Medical College, Philadelphia, PA). CD8 is a human T-lymphocyte surface antigen and was used to visually identify transfected cells using CD8 antibody–coated polystyrene microbeads. Chinese hamster ovary K1 (CHO-K1) cells were obtained from the American Type Culture Collection and maintained in HAMS F-12 media ( Gibco Laboratories ) supplemented with 1 mM l -glutamine, and 10% heat-inactivated fetal bovine serum ( Gibco Laboratories ) in a humidified, 5% CO 2 incubator at 37°C. CHO-K1 cells were cotransfected with the HERG and CD8 plasmids in a ratio of 4:1. Transfection was accomplished using the Lipofectamine transfection reagents and method ( Gibco Laboratories ). Immediately before patch clamping, cells were labeled with commercially prepared microbeads conjugated to CD8 antibodies (DynaBeads; Dynal ) to identify transfected cells. Cells that displayed CD8 on their surface bound DynaBeads, indicating successful transfection . The intracellular recording solution for all experiments was (mM): 110 KCl, 5 K 2 ATP, 5 K 4 BAPTA, 2 MgCl 2 , 10 HEPES, pH 7.2. The control extracellular recording solution was (mM): 145 NaCl, 4 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 HEPES, 10 glucose, pH 7.35. Solutions with elevated divalent cation concentrations were made by adding an appropriate amount of 1 M aqueous chloride salt solution to the control extracellular solution. The 2 mM EGTA extracellular solutions in Fig. 4 A was the same as control except that CaCl 2 and MgCl 2 were omitted and 2 mM EGTA was added. For the Ca 2+ dose response in Fig. 4 B, contaminant Ca 2+ was assumed to be 25 μM. MgCl 2 was omitted and, for Ca 2+ concentrations ≥100 μM, the appropriate concentration of Ca 2+ was added to the solution without a Ca 2+ buffering agent. At concentrations <100 μM, 1 mM EGTA was used to buffer the Ca 2+ . The amounts of Ca 2+ and chelator needed to obtain a desired concentration of free Ca 2+ were determined using Sliders MaxChelator software with the BERS.CCM constants (Chris Patton, Stanford University, Stanford, CA). The whole-cell patch clamp technique was used to assay HERG channel function . Cells were patch clamped 36–60 h after transfection at a temperature of 20–25°C. HERG currents in transiently transfected CHO-K1 cells are very stable. We observe no rundown during whole cell voltage clamp over the lifetime of the cell, which can be well over 1 h. All recordings were made using an Axopatch 200A patch clamp amplifier in conjunction with a Digidata 1200 interface ( Axon Instruments ). Patch pipettes were fabricated from capillary glass using a Flaming/Brown micropipette puller (P-97; Sutter Instruments, Co. ). Patch pipette resistances were 1–2 MΩ. Cell and pipette capacitances were nulled and series resistance was compensated (85– 95%) before recording. Data were acquired using pCLAMP programs (6.03; Axon Instruments ). Data were analyzed and plotted using a combination of pCLAMP, Origin (Microcal Software), and SigmaPlot 4.0 (Jandel Scientific). In all figures featuring raw current recordings, the bottom of the current scale bar indicates the zero current level. We adopted a simple multistate Markov kinetic model from the literature . Our purpose was not to uniquely identify a model of HERG K + currents, but to test whether a simple voltage shift in one or more parameters of the published model could account for the data. Multistate Markov gating models have been successfully used for modeling voltage-gated ion channels and interpreting experimental observations . In the model, only the O state conducts ionic current. C states are closed states primarily occupied at negative membrane potentials. The I state is a closed state primarily occupied during membrane depolarization. The transitions along the activation pathway (see Terminology ) are governed by voltage-dependent rate constants ( a 1 , a −1 , a 3 , and a −3 ) and voltage-independent rate constants ( a 2 and a −2 ). Transitions to and from the inactivated states are governed by two additional voltage-dependent rate constants ( a 4 and a −4 ) . The rate constants were limited to a maximum value at extreme membrane potentials by applying the following: a n = [(1/ a n ) + (1/ a max )] −1 , where a max (s −1 ) is the limiting value of the rate constant ( a 1max = 100, a 3max = 25, and a 4max = 400). The simulations were carried out using ModelMaker (Cherwell Scientific). The rate constants for the forward and reverse transitions were calculated according to Eqs. 1 and 2 . 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}a_{n}=a_{n}(0)exp\hspace{.167em}(z{\delta}eV/k_{B}T)\end{equation*}\end{document} 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}a_{-n}=a_{-n}(0)exp\hspace{.167em}[-z(1-{\delta})eV/k_{B}T],\end{equation*}\end{document} where a n and a −n represent the forward and reverse transition rate constants between the nth state and the n + 1 state in units of s −1 , k B = 1.3807 × 10 −23 V C K −1 (Boltzmann constant), T = 293.15°K, e = 1.6022 × 10 −19 C (electronic charge in Coulomb). Each rate constant is defined by two terms. One term is the rate constant in the absence of a membrane potential, [ a n (0) and a −n (0)], and the other term is membrane potential dependent [ z δ e V/( k B T )]. The effective charge on the “gate” is z , the fraction of the electrical field sensed is δ, and the membrane potential is V. The same equations with changes in a n (0) and z were used for the other rate constants. The parameters for each of the transition rate constant pairs were as follows: a 1 , a −1 :δ 1 = 0.40, z 1 = 1.80, a 1 (0) = 6.963 s −1 , a −1 (0) = 11.481 s −1 ; a 2 , a −2 :δ 2 = 0.00, z 2 = 0.00, a 2 (0) = 8.172 s −1 , a −2 (0) = 42.977 s −1 ; a 3 , a −3 :δ 3 = 0.40, z 3 = 1.98, a 3 (0) = 11.481 s −1 , a −3 (0) = 0.094 s −1 ; a 4 , a −4 :δ 4 = 0.5, z 4 = 0.82, a 4 (0) = 116.824 s −1 , a −4 (0) = 14.023 s −1 ; V = V clamp − V shift . V clamp is the voltage clamp potential applied. V shift is the apparent change in membrane potential caused by Ca 2+ binding and was set to zero except to simulate elevation of external Ca 2+ . We found that applying the same V shift to all rate constants could not simulate the observed data. A shift in a 4 and a −4 also altered simulated inactivation, inconsistent with our data. Therefore when simulating elevated Ca 2+ (10 mM) V shift for a 1 , a −1 , a 3 , and a −3 was set to +23 mV so that only the voltage dependence of the activation rate constants were affected, and V shift for a 4 and a −4 was set to 0 mV. Rate constants a 2 and a −2 are voltage independent. HERG K + currents measured with the voltage clamp protocols described in the results were used for adjusting the model parameters to approximate the data obtained in 1.8 mM Ca 2+ . The model K + currents were scaled to fit the data sets using a linear open channel current–voltage relationship and a K + channel reversal potential of −90 mV to correspond with the experimentally determined value. The HERG open channel current–voltage relationship has been described as linear in several studies . The simple model of HERG gating described above can be further simplified to facilitate the discussion of the experimental current measurements. There are three main classes of kinetic states needed for the discussion: closed, open, and inactivated states (Scheme II). C (closed) and I (inactivated) are both nonconducting states. Only the O (open) state conducts ionic current. Channels are primarily in closed states at negative voltages. Membrane depolarization predisposes the channels to move toward the open and inactivated states. Repolarization allows channels to return to the closed state. It is important to note that in this model, as in the more complicated model described above, inactivated channels may close only after recovering through the open state. HERG channel gating has been described as a depolarization-activated channel with slow activation, and fast inactivation gating (Scheme I), in accordance with the previous description of I Kr gating . However, it is also possible to describe this channel as a hyperpolarization-activated channel where channel activation is more rapid than inactivation . This distinction is semantic and we will discuss our data using the terminology indicated in the scheme above. Fig. 1 shows the effects of elevated external Ca 2+ on HERG channel currents. Increasing the external Ca 2+ concentration from 1.8 to 10 mM decreased the current amplitude and enhanced the rate of decay of tail currents at −50 mV . Plotting the maximal current during the test step versus the test potential results in the bell-shaped current–voltage relationship characteristic of HERG channels seen in Fig. 1 C . The decrease in the current–voltage relationship at positive potentials is due to voltage-dependent channel inactivation . Elevating the external Ca 2+ concentration increased the degree of depolarization required to activate HERG currents . However, at membrane potentials greater than +40 mV, current amplitudes during the test pulse were nearly the same in 1.8 or 10 mM Ca 2+ . Plotting the peak tail current amplitude measured at a constant potential (−50 mV) as a function of the preceding test potential resulted in a sigmoidal curve that described the voltage dependence of channel opening . Elevation of external Ca 2+ from 1.8 to 10 mM shifted the midpoint of this tail current curve by +22.3 ± 2.5 mV ( n = 3), in agreement with the shift in voltage dependence of currents measured during the test step . Ca 2+ also decreased the maximum tail current amplitude, but the slope factors of the tail current curves were not significantly different between 1.8 (8.8 ± 0.2 mV, n = 3) and 10 (9.0 ± 0.4 mV, n = 3) mM Ca 2+ . To understand the nature of these Ca 2+ effects, the changes in activation and deactivation kinetics were characterized in greater detail. Because HERG inactivates very rapidly (relative to activation) at most membrane potentials, the rate of activation cannot be observed accurately during a single voltage clamp step . The time course of the K + current observed during a test pulse reflects the rates of both activation and inactivation . To estimate the rate of channel opening at positive potentials, we took advantage of the channel's rapid recovery from inactivation (compared with deactivation). This allowed determination of the rate of activation using the method shown in Fig. 2 A . A variable duration prepulse is used to drive channels into the open and/or inactivated state before stepping to a negative potential (−120 mV), which allows rapid recovery from inactivation (see Scheme II, the current measured at −120 mV is inward since the K + reversal potential was −90 mV). Measuring the time course at different prepulse potentials allowed determination of the voltage dependence of the rate of activation. Comparison of these rates in 1.8 mM extracellular Ca 2+ and 10 mM Ca 2+ (D) shows that activation was slower in the presence of 10 mM Ca 2+ . Elevated Ca 2+ decreased the rate of HERG activation at all potentials measured (+60 to 0 mV). This can be seen in Fig. 2 B, which shows activation time constants determined by fitting a second order exponential equation to the data (see legend for details). The slow time constant of activation was significantly increased ( P < 0.05) by elevating extracellular Ca 2+ from 1.8 to 10 mM for all test potentials between +50 and 0 mV. The fast time constant of activation was significantly increased ( P < 0.05) for test potentials +50, +40, +30, and 0 mV. Deactivation kinetics were examined by measuring the voltage dependence of the time course of the K + current decay upon stepping to different membrane potentials as shown in Fig. 3 . Time constants of deactivation were determined by fitting a monoexponential equation to the initial current decay. The initial phase of deactivation is well fit by a monoexponential allowing a simple quantitation of the change in deactivation rate upon elevation of extracellular Ca 2+ . Time constants determined in this manner were plotted as a function of the test potential to obtain the curves in Fig. 3 D. Increasing external Ca 2+ from 1.8 to 10 mM increased the rate of channel deactivation at every potential measured ( P < 0.05), resulting in smaller measured time constants . Under physiological conditions, Ca 2+ is always present in the extracellular environment, and it is conceivable that extracellular Ca 2+ is required for gating . Voltage-dependent block of the open channel could be a mechanism of channel deactivation, as has been suggested for I K in rabbit sinoatrial node . Fig. 4 A shows that HERG channels continued to gate when bathed in a solution with no added Ca 2+ or Mg 2+ and buffered with 2 mM EGTA. Removal of extracellular divalent cations increased the K + current immediately upon solution exchange. Channels opened more rapidly upon membrane depolarization leading to a transient current peak that rapidly decays with the onset of inactivation. Channels also closed more slowly upon repolarization, but channels continued to gate. These effects were rapidly and completely reversible upon the return of control extracellular solutions. These results are consistent with the effects of raising extracellular Ca 2+ seen in Figs. 1 – 3 , where opposite effects are seen. Fig. 4 B shows the Ca 2+ concentration dependence of the time constant of deactivation at −120 mV. If channel closing results from block by external divalent cations, then gating should cease in the absence of divalent cations. This is not the case. Fig. 4 B shows that gating persists at extremely low extracellular Ca 2+ concentrations. In fact, the rate of deactivation approaches a limiting value (time constant ∼50 ms) for Ca 2+ concentrations below 100 μM. Thus, divalent cation pore block cannot be the mechanism of deactivation gating for HERG channels, nor are these ions absolutely required for gating. Fig. 1 indicates that raising extracellular Ca 2+ results in decreased outward K + currents. Fig. 4 shows that time-dependent block of the channel by divalent cations is not the mechanism of the channel deactivation since deactivation occurs even in the absence of Ca 2+ and Mg 2+ . To determine whether the Ca 2+ effect is due to a rapid voltage-dependent block of open HERG channels, the method of Gilly and Armstrong was modified to accommodate HERG inactivation behavior. The instantaneous current–voltage relationships were measured in 1.8 and 10 mM external Ca 2+ . Increasing external Ca 2+ from 1.8 to 10 mM did not alter the shape of the open channel current– voltage relationship. The K + current was decreased in 10 mM Ca 2+ by ∼20% at all potentials measured , indicating that the Ca 2+ effect was independent of membrane potential. This finding is not consistent with the idea that the positively charged Ca 2+ is acting as a rapid voltage-dependent blocker of the channel . Therefore, neither slow nor fast voltage-dependent block is the mechanism of the Ca 2+ effect. We next investigated the possibility that the apparent Ca 2+ -induced decrease in conductance was due to the observed changes in channel gating. Increasing extracellular Ca 2+ slowed the rate of HERG activation and accelerated deactivation . We therefore examined whether these kinetic changes could account for the Ca 2+ -induced reduction of HERG current seen in Fig. 5 C. The protocol for measurement of this instantaneous current–voltage relationship contains a 12.5-ms step to −100 mV that was required to allow the channels to recover from inactivation. Thus, in elevated Ca 2+ , accelerated deactivation might result in more channels deactivating during the 12.5-ms recovery step, before the measurement of current. This would result in fewer open channels at the time of the test step and hence would reduce the instantaneous K + current measured upon stepping to +30 mV. Therefore, we measured the rate of deactivation at −100 mV and used this information to estimate the deactivation that occurred before measurement. This estimation was used to scale instantaneous current–voltage curves for the deactivation that occurs during the 12.5-ms recovery step at −100 mV . Exact determination of the degree of deactivation that occurs during the recovery step is not possible if channels must recover from inactivation before deactivating (Schemes SI and SII ). Nonetheless, because deactivation at −100 mV is more than an order of magnitude slower than recovery from inactivation , it is possible to estimate the fraction of channels that have deactivated during the 12.5-ms recovery step using the method in Fig. 5 . When the enhanced rate of deactivation induced by 10 mM Ca 2+ was accounted for, the apparent decrease in current was eliminated . This resulted in instantaneous current–voltage relationships in 1.8 and 10 mM Ca 2+ that superimpose. This analysis illustrates that the reduction of HERG K + current observed in elevated extracellular Ca 2+ concentrations results from the kinetic changes caused by Ca 2+ . With this information on the influence of deactivation on the instantaneous current–voltage curves in Fig. 5 C, the decrease in outward current seen in the isochronal activation curve with 10 mM Ca 2+ in Fig. 1 D was reexamined. To test whether this decrease in current might also be due to the observed changes in gating kinetics, a three-pulse protocol was used to measure the voltage dependence of isochronal activation . Deactivation at −100 mV was measured in each test solution immediately after measurement of the voltage dependence of isochronal activation in Fig. 6 B. The activation curves observed with the three-pulse protocol and the curves scaled to account for deactivation are shown in Fig. 6 , B and C, respectively. This scaling for deactivation removed the observed reduction in HERG peak tail currents, leaving only a parallel shift of the curve to more positive membrane potentials. If the observed reduction of K + current results from enhanced deactivation of the channels before measurement, then the decrease should become greater if a longer recovery period allows the degree of deactivation to increase. We analyzed data from protocols using 12.5-, 25-, and 50-ms recovery periods as an internal control for this method of analysis (data not shown). Increasing recovery duration results in progressive decreases in measured current (due to increasing contamination from deactivation). Likewise, increasing the length of recovery increases the amount of current suppression by addition of 10 mM Ca 2+ . In each case, this decrease could be removed by accounting for deactivation. This analysis illustrates that the reduction in observed outward current in 10 mM Ca 2+ is not due to channel block by Ca 2+ . The data in Figs. 4 and 5 also demonstrate that Ca 2+ does not block HERG directly. Instead, Ca 2+ appears to interact with or influence the channel voltage sensor(s) and alter the apparent membrane potential sensed by the channel gating machinery. A common way in which multivalent cations have been found to influence ion channel gating is through screening of diffuse negative surface charges . This can cause shifts in the membrane potential sensed by a membrane protein, such as a voltage-gated K + channel. This type of nonspecific surface charge screening should affect all channel parameters that depend on membrane potential . To determine whether all voltage-dependent channel parameters are equally affected by external Ca 2+ , the inactivation of HERG currents in the presence of 1.8 and 10 mM Ca 2+ was examined. The rate of channel inactivation was measured in 1.8 and 10 mM external Ca 2+ over a range of membrane potentials and under conditions designed to isolate the inactivation process. Two voltage-clamp protocols were used to determine the voltage dependence of inactivation and recovery from inactivation . A three-pulse protocol was used to measure the rate of inactivation at membrane potentials more positive than −60 mV . Monoexponential curves were fit to the rapidly declining current during the step to the test potential, and the time constants obtained from these fits are plotted versus membrane potential in Fig. 7 C (circles, right side). Fig. 7 C shows that, surprisingly, the voltage-dependent rate of inactivation was almost unaffected by elevation of extracellular Ca 2+ from 1.8 to 10 mM. The large shift in voltage-dependent gating behavior observed for activation and deactivation was not present. At membrane potentials more negative than −60 mV, it was difficult to accurately measure inactivation by the three step method described above. Therefore, a two-pulse protocol, described in Fig. 7 A, was used to measure the rate of recovery from inactivation between −130 and −50 mV. In this case, the channels were activated and inactivated by a 2-s pulse to +50 mV. Then, stepping to the test potential results in a rising phase of the tail current that is largely due to recovery from inactivation . The rate of recovery was estimated by fitting a monoexponential function to the rising recovery phase, and the time constant obtained was plotted as a function of the test potential in Fig. 7 C (squares, left side of curve) along with the time constants obtained for inactivation with the three-pulse protocol . As observed for the rate of inactivation, the rate of recovery from the inactivated to the open state was largely unaffected by increasing the Ca 2+ concentration to 10 mM. These findings support the hypothesis that there are separate voltage sensors for channel activation and inactivation gating and indicate that external Ca 2+ affects them differently. The very slow activating and fast inactivating phenotype of HERG currents prevents traditional determination of a steady state inactivation (h ∞ ) curve . The voltage dependence of the distribution of channels between the open and inactivated states can be tested by the method illustrated in Fig. 8 . A three-pulse protocol was used and the instantaneous current measured in the third step to +30 mV was plotted as a function of the test membrane potential . Note that these curves largely superimpose except at potentials less than −80 mV, indicating that 10 mM Ca 2+ had no effect on the voltage dependence of this relationship. The decrease in K + current observed for test steps more negative than −100 mV is again due to channel deactivation at these membrane potentials. Accounting for the enhanced rate of deactivation eliminates this decrease . Correction was accomplished in the same manner as was described for Figs. 5 and 6 , with the exception that the fraction of current lost due to deactivation must be determined for each test potential. Thus, both the corrected and uncorrected data indicate that Ca 2+ affects only deactivation and does not affect the voltage dependence of inactivation. A decrease in current measured with this voltage clamp protocol due to accelerated deactivation is also predicted by the model discussed below. To determine whether changes in external Ca 2+ near the physiological range impact HERG K + currents during the time course of a cardiac action potential, we used a voltage-clamp protocol with the shape of an action potential . A similar protocol has been used to study HERG current stably expressed in HEK-293 cells . Human total serum Ca 2+ concentrations range between 1.5 and 4.25 mM (average ∼2.5 mM), ∼50% of which is in the free ionized form . Fig. 9 illustrates how Ca 2+ modulation of HERG gating results in changes in current during an action potential clamp. The action potential clamp protocol was applied to a cell expressing HERG K + channels in solutions containing 1, 2, 3, or 10 mM external Ca 2+ . Relatively little outward K + current was apparent during the peak of the action potential clamp, but as the cell membrane potential decreases (at the end of the action potential), HERG current rises sharply. HERG currents during this voltage clamp protocol were highly sensitive to changes in extracellular Ca 2+ concentration between 1 and 3 mM. Note that, although our experiments were at room temperature, the action potential duration used (∼400 ms) is appropriate for a human ventricular myocyte at 37°C. As a consequence, these experiments may underestimate the impact of Ca 2+ on the currents during an actual action potential. Changing external Ca 2+ from 1 to 3 mM resulted in a 50% reduction of the maximum outward K + current during this action potential voltage clamp protocol. For comparison with the concentrations used in the biophysical characterization of the Ca 2+ effect, 10 mM Ca 2+ was applied to the cell and caused an 80% reduction of the maximum current under these conditions. Our data suggest an intimate association of Ca 2+ with the HERG channel protein that alters the voltage dependence and kinetics of channel activation gating, while having little effect on inactivation gating. Figs. 1 – 3 show that increasing extracellular Ca 2+ from 1.8 to 10 mM had at least two effects on HERG currents. The voltage dependence of channel activation was shifted to more depolarized membrane potentials and the current amplitude was decreased at membrane potentials less than +50 mV. Several simple mechanisms could potentially explain these effects of Ca 2+ . First, voltage-dependent block of the channel might decrease HERG current at potentials less than +50 mV, producing an apparent shift in the voltage-dependent activation of the channels. Relief of block at positive potentials could then account for the lack of block at potentials +50 mV. This possibility was ruled out by two interdependent findings. Removing external divalents does not remove deactivation , eliminating the possibility that slow voltage-dependent block by divalent cations causes HERG deactivation. It is impossible to assure that all divalent cations have been removed from the external solution, but by omitting them from the solution and adding the chelator EGTA, we can assure that the concentration is extremely low (∼2.5 nM). We also showed that the time constant of HERG deactivation reaches a maximum (∼50 ms) in 100 μM Ca 2+ and does not increase for lower concentrations of Ca 2+ over five orders of magnitude. The shape of the open channel current–voltage relationship is not changed by elevating external Ca 2+ ; a voltage-dependent open channel blocker would change the shape of this curve . A second possible mechanism for Ca 2+ modulation is through charge screening of negative membrane surface charges, resulting in a change in the membrane potential sensed by the channel . Such a screening could be nonspecific, resulting from association with diffuse charged moieties like membrane phospholipid head groups, or it could occur through specific interaction with the channel protein. Nonspecific surface charge screening would cause a global change in the membrane potential (V) perceived by the entire channel protein, and therefore all voltage-dependent parameters of the channel would be shifted in a parallel manner along the voltage axis by the change in perceived membrane potential (V = V clamp − V shift ). The fact that the voltage dependence of activation gating was shifted by elevated external Ca 2+ while inactivation gating was not indicates that nonspecific charge screening inducing a change in the membrane potential sensed by the entire channel cannot be the mechanism of Ca 2+ modulation. Ca 2+ association with a specific channel residue(s) could cause more selective effects on channel function since it need not result in a global change in the potential sensed by the channel protein. The observation of differential modulation of the voltage dependence of activation and inactivation greatly constrains the possibilities for the nature of the channel voltage sensing machinery. Both HERG activation and inactivation gating appear voltage dependent . However, it is possible that inactivation derives its apparent voltage dependence from coupling to voltage-dependent channel activation, as is true for Shaker potassium channels . In this case, any effect on the activation voltage sensor would also alter the apparent voltage dependence of inactivation, contrary to our data and previous observations of K + modulation of HERG . Alternatively, both processes could be intrinsically voltage dependent, but use the same voltage-sensing machinery. This possibility is also excluded by the specificity of the Ca 2+ effect. If the same voltage sensor controlled both activation and inactivation, Ca 2+ shielding of that sensor should affect both gating processes, contradicting the data. Our data seem to indicate that there are at least two voltage-sensing regions involved in HERG channel gating: one governing channel activation and another for inactivation. Furthermore, these distinct sensors can detect different local values of membrane potential and be differentially screened by Ca 2+ . To test the feasibility of this idea, we used a simplified HERG channel gating model to determine whether our observations could be predicted by a voltage shift in only the voltage dependence of the rate constants that govern channel activation (see methods for details). The model simulates the major features of our own data and is consistent with data from the literature . In the model, both activation and inactivation are voltage dependent . Fig. 10 A illustrates simulated currents for the voltage-clamp protocol in Fig. 1 . Elevated Ca 2+ was simulated by including a +23-mV bias in the rate equations for the activation rate constants ( a 1 , a −1 , a 3 , a −3 ). As seen in Fig. 10 , this change alone accounts for a decrease in K + currents and an increase in the rate of deactivation . Fig. 10 C plots the simulated outward current after 2 s at the test potential, and D shows the peak tail current . Fig. 10 E indicates that the simple shift of voltage-dependent activation rate constants in the model also results in an apparent decrease in the open channel current–voltage relationship . Furthermore, the modeled voltage shift does not affect the voltage dependence of the equilibrium between the open and inactivated states . The model also predicts the Ca 2+ -induced decrease in K + current measured at negative test potentials with this protocol . This occurs because the rate of deactivation is accelerated in elevated extracellular Ca 2+ . Therefore, at test potentials more negative than the threshold for channel activation, the faster deactivation in 10 mM extracellular Ca 2+ allows more channels to make the transition from the open to the closed state. As a result, 10 mM Ca 2+ exaggerates the decrease in the channel availability curve measured at negative potentials in Fig. 8 B, as is simulated in the modeled data . Thus, a simple shift in the membrane potential felt by the activation process, but not the inactivation process, explains the effects experimentally observed upon elevation of extracellular Ca 2+ . Such an effect can be envisioned if we assume Ca 2+ binds to a region of the HERG protein near the region that controls activation voltage sensing and alters the membrane potential sensed by amino acids in the vicinity of the binding site. Voltage shifts applied to both activation and inactivation rate constants in the model failed to qualitatively reproduce the experimental observations (not shown). Although we cannot definitively rule out the possibility that Ca 2+ causes an allosteric change in HERG channels that mimics a specific association near the activation voltage sensor, our data are consistent with a simple Ca 2+ -induced change in membrane potential sensed specifically by the activation process. Ca 2+ is critical in the gating of some voltage-gated channels. HERG belongs to the family of six transmembrane domain voltage-gated K + channel genes such as Shaker , EAG , and the mammalian Kv channels. The effects of Ca 2+ have been most thoroughly characterized in the squid delayed rectifier K + channel and the recombinant Shaker K + channel; both require divalent cations to stabilize the closed channel and maintain channel selectivity . Unlike these channels, we find that HERG channels do not absolutely require Ca 2+ for selectivity and gating, though even control extracellular Ca 2+ concentrations cause a shift relative to the gating behavior in their absence . These results agree with data for I Kr recorded from guinea pig ventricular myocytes in high K + . Our data show that the voltage dependence of the HERG activation curve was shifted by +22.3 ± 2.5 mV upon raising extracellular Ca 2+ from 1.8 to 10 mM. This shift was accompanied by slowed channel activation, and enhancement of the rate of channel deactivation, even in physiological concentrations of external permeant cations (4 mM K + ). Deactivation of I K in rabbit sinoatrial node myocytes is sensitive to external divalent cations, and block by the extracellular divalent cations Ca 2+ and Mg 2+ has been proposed to be the primary determinant of voltage-dependent deactivation gating of that current . Our data indicate that HERG K + channels retain deactivation gating even in the absence of external divalent cations . Furthermore, the apparent decrease of current through open HERG channels was not voltage dependent . This indicates that Ca 2+ does not experience the transmembrane field when interacting with HERG channels. Thus, neither a time-dependent nor -independent voltage-dependent block of open HERG channels can explain the effect of extracellular Ca 2+ . The observed decrease in HERG K + currents upon elevation of external Ca 2+ was completely explained by modulation of gating. Correction of the current amplitudes for these Ca 2+ -induced changes in gating removed the apparent current suppression . Thus, external divalent cations modulate, but do not cause, HERG deactivation gating. This is in contrast with the conclusions that divalent cations cause deactivation gating of rabbit I K . There are several possible explanations for this discrepancy. It is conceivable that native rabbit sinoatrial node K + channels interact with divalent cations in a fundamentally different manner than heterologously expressed HERG channels due to differences in structure or subunit composition. However, there is considerable similarity in the behavior of rabbit sinoatrial node I K and HERG . A more likely explanation involves the differences in the external ionic conditions between the two studies. Our study was performed in 4 mM external K + , while studies in rabbit sinoatrial node were performed in the presence of high extracellular K + (140 mM). Elevated external K + was necessary in part because the native currents are small in physiological K + . Both HERG K + currents and I Kr are modulated by external K + . Elevated extracellular K + slows inactivation and deactivation gating of HERG. Slow deactivation kinetics induced by high K + could make deactivation in the absence of extracellular divalent cations difficult to observe. This difficulty is exacerbated by the apparent competition between external K + and Ca 2+ . Elevated extracellular K + (20 mM) decreases the shift in the HERG channel isochronal current–voltage curve caused by Ca 2+ . This functional interaction was interpreted as a competition for a binding site within the channel pore . Since our data indicate that Ca 2+ does not directly occlude the permeation pathway of HERG channels, this competition between external K + and Ca 2+ is most likely at a site outside the pore, or it may be a functional result of the two ions exerting antagonistic effects on channel gating at distinct sites. The specificity of Ca 2+ for HERG activation gating is further evidenced by the identity of K + current at membrane potentials +10 mV in Fig. 1 C. This lack of current suppression could be interpreted as relief of Ca 2+ block by positive membrane potentials . However, Figs. 4 and 5 illustrate that Ca 2+ is not a voltage-dependent blocker of HERG currents. HERG outward currents are determined by both activation and inactivation controlling channel open probability. Outward currents during depolarization to intermediate potentials are suppressed because a shift of the voltage dependence of channel opening to more positive potentials leads to a decrease in channel open probability . At membrane potentials greater than +10 mV, activation approaches a maximal level, and therefore channel inactivation is the primary determinant of HERG channel open probability at these voltages. HERG inactivation is much less sensitive to external Ca 2+ than is activation . Therefore at potentials greater than +10 mV, where inactivation largely determines HERG open probability, there is no Ca 2+ -induced decrease in current. Still another illustration of the specificity of the Ca 2+ effect on activation gating is seen in Fig. 4 A. The current recorded in divalent cation–free solution showed a distinct inactivating phase during depolarization to +50 mV. This is because the rate of activation was increased upon removing Ca 2+ . Increased Ca 2+ slowed activation , but complete removal of divalent cations increased the rate of activation, while the rate of inactivation was little affected by changes in external Ca 2+ . The result is that a significant fraction of the channels were activated before the onset of inactivation, and a portion of the current inactivation could therefore be directly observed . It has been suggested that the inactivation of HERG K + channels is mechanistically related to the C-type inactivation identified in Shaker K + channels . Indeed, HERG inactivation shares several characteristics with C-type inactivation. For example, the rate of HERG inactivation is sensitive to extracellular K + and TEA, but insensitive to intracellular TEA . The rate and voltage dependence of HERG inactivation are also dependent upon the identity of the amino acid present at position 631, analogous to Shaker residue 449, which in part determines the rate of C-type inactivation in those channels . Unlike C-type inactivation in Shaker channels, HERG inactivation occurs more rapidly than activation at some potentials and has been suggested to have its own independent voltage sensor . Our data support this conclusion. Neither N- nor C-type inactivation of Shaker K + channels have significant inherent voltage sensitivity. The apparent voltage dependence at positive potentials occurs through coupling to the voltage dependence of activation gating . However, it is now clear that, for HERG channels, mutation of serine 631 to alanine causes a shift in the voltage dependence of channel inactivation without shifting the voltage dependence of activation . The selectivity of the effect of Ca 2+ on the activation gating process provides further support for the idea that HERG-type inactivation has a distinct voltage sensor and is therefore fundamentally different from C-type inactivation in Shaker -like channels. Fig. 9 shows that raising external Ca 2+ from 1 to 3 mM reduced the HERG current maximum by 50% during an action potential voltage clamp. This large effect may seem surprising, but it is actually quite consistent with the biophysical characterization of the Ca 2+ effect. The characteristic slow activating, fast inactivating behavior of HERG channels results in outward currents during the action potential clamp that are largest near the midpoint of the voltage-dependent activation curve. Hence, Ca 2+ -induced shifts in the midpoint of the activation curve greatly affect the current level during an action potential clamp. HERG currents recorded with a voltage clamp protocol of this type at 35°C display more current during the plateau phase of the action potential waveform due to the temperature dependence of the activation process . As a result, calcium effects could be even larger at physiological temperatures than they are in our study. Our results demonstrate how even modest changes in external Ca 2+ concentration could significantly change the fraction of HERG channels available to participate in cellular repolarization in vivo. We conclude that elevation of extracellular Ca 2+ interacts specifically with channel activation voltage sensing, and does not act as a direct blocker of the open pore. This interaction is likely mediated by the association of Ca 2+ with distinct, possibly negatively charged, channel residues near a voltage sensor. Voltage-dependent inactivation gating was nearly unaffected by elevating external Ca 2+ . The differential effect of external Ca 2+ on the voltage dependence of activation and inactivation indicates that these two processes sense different values of transmembrane potential and are therefore probably physically separated in space. Further studies will be needed to elucidate the specific channel residues that are responsible for this interaction. | Study | biomedical | en | 0.999997 |
10102938 | Due to their primary importance in both initiating and modulating repolarization of the cardiac action potential, extensive experimental effort has been devoted to analysis of potassium channels as potential targets for antiarrhythmic agents . In particular, the calcium-independent transient outward potassium current I to has recently received much experimental attention. I to is present in nearly all mammalian working cardiac myocyte types, including both human atrial and ventricular myocytes . Due to its rapid activation kinetics, I to is the major current responsible for the early “notch” or phase 1 repolarization characteristic of the mammalian ventricular action potential. In addition, due to its slower inactivation kinetics, I to can also significantly modulate the plateau and early phase 3 repolarization . Despite the nearly universal presence of I to in mammalian working myocardium, it appears to be less widely appreciated that there may be multiple and functionally distinct I to phenotypes. In particular, significant differences in specific gating characteristics of I to phenotypes have been reported both among different species and within different cardiac tissue and myocyte types from the same species. Three major differences can be summarized as follows: (a) marked differences in activation thresholds exist , (b) the kinetics of macroscopic inactivation are either clearly single exponential or markedly biexponential , and (c) the kinetics of recovery from inactivation are either rapid or slow to very slow . Hence, there is a 1–2-order-of-magnitude difference in the time constants of recovery reported for different cardiac I to phenotypes. Due to these marked differences in recovery kinetics, very slowly recovering cardiac I to phenotypes display cumulative inactivation during rapid and repetitive voltage clamp protocols , while rapidly recovering phenotypes generally do not . In addition to significant kinetic differences among I to phenotypes, within any one given species there may also exist heterogeneous expression of multiple I to phenotypes and/or I to current densities among distinct anatomical regions of the heart . For example, recent studies on human left ventricular epicardial and endocardial myocytes have indicated a marked difference in both I to current density and kinetics of recovery from inactivation between the two myocyte types . Thus, depending on both species and specific anatomical region, there appear to be at least two major I to phenotypes in mammalian heart capable of exerting functionally distinct frequency-dependent modulatory effects on repolarization. The presence of at least two functional I to phenotypes suggests the existence of at least two distinct potassium channel α subunits underlying each of the currents. To date, two main approaches have been used to address the issue of which α subunit underlies cardiac I to : (a) detection of α subunit mRNA levels (generally of whole cardiac tissue samples), and (b) comparisons of kinetic characteristics of heterologously expressed potassium channel α subunit clones to native myocyte I to phenotypes measured in one particular species. Based on these approaches, Kv1.4, Kv4.2, and Kv4.3 (long and short isoforms) α subunits have all been recently proposed to underlie “cardiac I to ” . However, previous studies have not adequately examined the spatial distribution of both multiple I to phenotypes and/or heterogeneity of Kv α subunit transcript and protein expression . Given these limitations, it is important to correlate levels of expressed I to channel α subunit proteins in specific anatomical regions of the heart to patterns of functional I to current phenotypes measured from individual myocytes isolated from the same anatomical regions. To begin to address these issues, in this study we first demonstrate, using whole cell patch clamp, that at least two kinetically and pharmacologically distinct I to phenotypes exist in ferret isolated left ventricular epicardial (LV epi) 1 and endocardial (LV endo) myocytes. We designate these two major I to phenotypes as I to,epi and I to,endo . We then demonstrate, using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence (IF) techniques, that Kv1.4, Kv4.2, and Kv4.3 α subunit proteins are all expressed in ferret isolated LV myocytes. However, we show that the specific distribution patterns of these three candidate I to α subunits are quite heterogeneous among LV epi versus LV endo myocytes: (a) both Kv4.2 and Kv4.3 are each more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 is expressed in the majority of LV endo myocytes but is essentially absent in LV epi myocytes. These combined patch clamp and FISH/IF results on isolated myocytes therefore strongly suggest that in ferret heart Kv4.2/Kv4.3 and Kv1.4 α subunits, respectively, are the underlying molecular substrates for the I to,epi and I to,endo phenotypes. We then extend the FISH and IF measurements to sagittal ventricular tissue sections so as to gain initial insights into overall distribution patterns of Kv1.4, Kv4.2, and Kv4.3 I to α subunits in the whole ventricle. We demonstrate that (a) Kv4.2 is predominantly expressed in LV apical epicardium, (b) Kv1.4 is predominantly expressed in LV apical endocardium, and (c) Kv4.3 is relatively more uniformly expressed throughout the LV wall. Hence, in the intact left ventricle, Kv1.4 and Kv4.2 expression levels vary significantly in the LV wall both transversely (i.e., epicardium-to-endocardium) and sagittally (i.e., apex-to-base). In contrast, in right ventricular (RV) tissue, we demonstrate that Kv4.2 is uniformly expressed across the entire free wall, while Kv1.4 and Kv4.3 are relatively sparse to absent. This latter result may imply that Kv4.2 α subunits underlie the much more uniformly expressed I to phenotype in ferret right ventricular myocytes . Hearts were obtained from 16–30-wk-old male ferrets. Depending on photo period and other environmental factors, ferrets can reach sexual maturity at ∼16–32 wk ; hence, potential developmental changes in potassium channel expression occurring during puberty among some of the ferrets used cannot be definitively ruled out. Myocytes were enzymatically isolated (collagenase, protease, elastase) from the left ventricular free wall via a Langendorff perfusion apparatus exactly as described , except for the following slight modification. After 5–10 min of initial enzyme perfusion, the heart was removed from the Langendorff apparatus and the free wall of the left ventricle was dissected from the adjacent apical, septal, and basal regions, resulting in a tissue sample corresponding approximately to the middle one- to two-thirds of the left ventricular wall. Small thin strips (∼1 mm thick × 0.5–1 cm long) were then dissected in a sagittal orientation from both the partially digested epicardial and endocardial surfaces. These epicardial and endocardial tissue strips were then separately reincubated in fresh enzyme solution, gently shaken at 37°C, and myocytes were harvested at 10–20-min intervals as previously described . Once isolated, myocytes were directly stored in normal control Na + - and Ca 2+ -containing saline (see below) at room temperature (21–22°C). All voltage clamp experiments were conducted on myocytes within 6–12 h of isolation. All patch clamp recordings were conducted at room temperature (21–22°C) using the whole cell ruptured patch configuration . Exactly the same equipment, perfusion system, glass tubing for patch pipettes, etc., as previously described in detail were used in the present study. Data were recorded (filtered at 1–2 kHz) on video tape, and then subsequently digitized (5–10 kHz) off-line and analyzed using pClamp and FigP software . Data are summarized as mean ± SEM. After attainment of the whole cell configuration in normal control saline (mM: 144 NaCl, 5.4 KCl, 1 MgSO 4 , 1.8 CaCl 2 , 10 HEPES, pH 7.40), I to was isolated from other overlapping currents by perfusing the myocytes with Na + - and Ca 2+ -free extracellular “I to saline” (mM: 144 N -methyl- d -glucamine, 5.4 KCl, 2.3 MgSO 4 , 0.5 CdCl 2 , 10 HEPES, pH 7.40). Patch pipettes (3–4.5 MΩ) contained (mM): 140 KCl, 1 MgSO 4 , 5 Mg-ATP, 5 Tris-creatine phosphate, 0.2 GTP, 5 EGTA, 10 HEPES, pH 7.40. We opted for 500 μM Cd 2+ in the extracellular I to solution to block the L-type calcium current, I Ca,L , for two reasons: (a) many commonly used organic I Ca,L blockers can exert significant nonspecific blocking effects on cardiac I to phenotypes , thereby potentially complicating analysis (e.g., use-dependent effects); and (b) for comparative purposes to previous I to studies that also used Cd 2+ to block I Ca,L . Cd 2+ can exert effects on various potassium currents, including shifts in both activation and inactivation, and such presently uncharacterized effects may not be identical among the two major I to phenotypes (I to,epi , I to,endo ) that we describe. The standard holding potential under all conditions was −70 mV. 5–15 min were routinely allowed to pass after initial attainment of the whole-cell configuration to allow for stabilization of currents and gating parameters . “Leakage correction” was not applied. All chemicals used for making solutions were obtained from Sigma Chemical Co. Heteropoda toxin 2 (stored at −20°C; directly added to room temperature I to saline at a final concentration of 150 nM immediately before experimental application) was a kind gift of NPS Pharmaceuticals. Kv1.4 (monoclonal), Kv4.2 (COOH-terminal polyclonal), and Kv4.3 (COOH-terminal polyclonal) antibodies were prepared as follows. The anti–Kv1.4 monoclonal antibody K13/31 was raised against a synthetic peptide (NSHMPYGYAAQARARERERLAHSR; Quality Controlled Biochemical ) corresponding to amino acids 13–37 of rat Kv1.4. This sequence is 100% identical to the corresponding sequence of ferret Kv1.4 . The anti–Kv4.2 polyclonal antibody Kv4.2C was raised against a synthetic peptide (CLEKTTNHEFVDEQVFEES; Quality Controlled Biochemical ) corresponding to amino acids 484–502 of rat Kv4.2. The sequence of the corresponding region of ferret Kv4.2 was identical to that of rat Kv4.2 (M.J. Morales, unpublished observations). The anti–Kv4.3 polyclonal antibody Kv4.3C was raised against a synthetic peptide (SPGPNTNIPSITSN; Protein Chemistry Laboratory, Washington University Medical Center, St. Louis, MO) that matches a unique sequence in the COOH terminus of Kv4.3 that corresponds to amino acids 616–629 of rat Kv4.3 short form and 635–648 of rat Kv4.3 long form. This amino acid sequence is down stream from the 19 amino acid insert in the COOH terminus in the long form of Kv4.3 . This suggests that the Kv4.3 antibody detects both the short and long forms of the Kv4.3α subunits. A cysteine residue was added to the NH 2 terminus of each peptide to allow coupling to the keyhole limpet hemocyanin carrier protein, and the coupled peptides were sent to Caltag Laboratories for injection into rabbits. Sera were screened using ELISA assays and the antibody was affinity purified using the Immunopure Antigen/Antibody Immobilization Kit #2 ( Pierce Chemical Co. ). The specificity of the Kv1.4 antibody was established in a previous report . The specificity of the Kv4.2 and Kv4.3 antibodies was examined in immunohistochemical experiments on Xenopus oocytes expressing Kv4.2 and Kv4.3 channels. The Kv4.2 and Kv4.3 (short form) cDNAs were obtained from Lily Jan (University of California, San Francisco, San Francisco, CA) and Jane Dixon and David McKinnon (SUNY, Stony Brook), respectively. Messenger RNA (Kv4.2, Kv4.3) or distilled H 2 O was injected into Xenopus oocytes and incubated for 72 h at 22°C in antibiotic containing Barth's solution as previously described . Two electrode voltage-clamp analysis was performed to document the presence of expressed Kv4.2 and Kv4.3 channels. Oocytes expressing Kv4.2 channels were tested with Kv4.2 and Kv4.3 antibodies, and cross-reactivity was assessed by preabsorbing the potential epitopes with Kv4.3 antibody and subsequently incubating with fluorescently labeled anti–Kv4.2 antibody. Similarly, oocytes expressing Kv4.3 channels were tested with Kv4.3 and Kv4.2 antibodies, and cross-reactivity was assessed by preabsorbing the potential epitopes with Kv4.2 antibody and subsequently incubating with fluorescently labeled anti–Kv4.3 antibody. Immunofluorescence on oocytes was performed as follows: oocytes were incubated in blocking buffer containing 5% BSA in TBSN (Tris buffered saline with NP40; 155 mM NaCl, 10mM Tris-Cl, pH 7.4, and 0.1% NP40) for 10–16 h at 4°C, and then washed 3 × 5 min in TBSN at room temperature. Oocytes expressing Kv4.2 and Kv4.3 were incubated with Kv4.2- and Kv4.3-specific primary antibodies (1:100), respectively, diluted in blocking buffer (indirect IF assay); another set of Kv4.2- and Kv4.3-expressing oocytes were separately incubated with anti–Kv4.2 and –Kv4.3 antibodies (1:100) diluted in blocking buffer as a preabsorption step. Both of these incubations were carried out at 4°C for 10–16 h. The oocytes were washed 5 × 5 min in TBSN. The first set of oocytes incubated with the respective primary antibodies were subsequently incubated with the secondary antibody, anti–rabbit IgG (1:200) conjugated with FITC at room temperature for 6 h. A second set of oocytes preabsorbed with anti–Kv4.2 or –Kv4.3 antibody were incubated with FITC-labeled anti–Kv4.2 antibody (Kv4.2-expressing oocytes) or FITC-labeled anti–Kv4.3 antibody (Kv4.3-expressing oocytes) for 10–16 h in the dark at 4°C (direct IF assay). The oocytes were then washed 5 × 10 min in TBSN, dehydrated in 100% methanol, and subsequently treated with BA:BB (one part benzyl alcohol to two parts benzyl benzoate) cleaning solution, mounted, and scanned using a confocal microscope (see below). Cardiac membrane proteins were prepared using a slight modification of the protocol previously described by Barry et al. . In brief, protein preparations were obtained from strips of tissue dissected from ferret LV epicardial and endocardial regions (exactly as described above for myocyte isolation). All procedures were conducted at 4°C and all solutions contained the following protease inhibitors: iodoacetamide (0.6 mM), 1,10 phenanthroline (0.5 mM), benzamidine (0.5 mM), leupeptin (0.15 μM), pefebloc (0.5 mM), aprotinin (2 μg/ml), and pepstatin (1 μM). The tissue strips were homogenized using a polytron (Brinkmann Instruments, Inc.) in 10 vol of 0.25 M sucrose buffer (whole tissue homogenate), followed by centrifugation at 1,075 g for 10 min (to remove nuclei and cellular debris). The pellet was used to prepare cytosolic- and particulate-enriched fractions as described by Storrie and Madden and Dignam . The supernatant was centrifuged at 105,000 g for 1 h at 4°C. The crude membrane pellet was suspended in Tris-EDTA (TE) buffer, and then centrifuged at 60,000 g for 30 min. The pellets were suspended in TE buffer containing 0.6 M KI, incubated on ice for 15 min, and then centrifuged again at 60,000 g for 30 min, washed once with TE buffer and centrifuged at 60,000 g . The pellets were resuspended in TE buffer containing 2% deoxycholate and incubated on ice for 1 h. Insoluble materials were removed by centrifugation at 13,175 g for 20 min and the supernatant was aliquoted and used for immunoblot analysis. Protein concentration was measured using a standard BCA method ( Pierce Chemical Co. ) and 50 μg of membrane protein with appropriate protein markers were run on SDS PAGE gel (10%), followed by transfer to Hybond-P PVDF membrane ( Amersham Corp. ). After incubation with PBS-T (0.1% Tween20 in PBS) buffer containing 10% nonfat dry milk powder, the membranes were washed once in PBS-T for 5 min and incubated with appropriate primary antibodies (anti–Kv1.4, 4.2, and 4.3 antibodies). After incubation, the membranes were washed 3 × 5 min and incubated in horseradish peroxidase–conjugated secondary antibody (anti–mouse or –rabbit IgG). Then, the membranes were washed 5 × 10 min, incubated in ECL solution ( Amersham Corp. ), exposed to film and developed. Techniques for FISH were performed essentially as previously described by Brahmajothi et al. . Ferret hearts were perfused (Langendorff apparatus) with 4% paraformaldehyde in PBS. Regions of interest were postfixed, incubated in 40% sucrose in PBS, and subsequently embedded in ornithine carbamyl transferase medium and frozen. Blocks were sectioned using a cryotome at a thickness of 6–7 μm and laid out on gelatin-coated slides. The sections were then postfixed in paraformaldehyde and incubated in prehybridization and hybridization buffer with specific sense and antisense oligonucleotide probes labeled with biotin or digoxigenin. The sections were incubated at 42°C for 16–24 h. After hybridization washes, detection of transcript was carried out using streptavidin–phycoerythrin or antidigoxigenin antibody conjugated with FITC. Hybridization signals from troponin I cardiac (TnIC) antisense probe was used as positive control and TnIC, Kv1.4, Kv4.2, and Kv4.3 sense probes were used as negative controls. The fluorescent signals were scanned using an inverted confocal microscope (LSM 410; Carl Zeiss, Inc. ) as detailed below. Techniques for IF were performed essentially as described by Brahmajothi et al. . Postfixed cryosections were initially blocked with 10% goat serum, washed once with PBS, and incubated with anti–Kv1.4 (1:1,500), anti–Kv4.2 (1:100), and anti– Kv4.3 (1:50) antibodies for 1 h, and then washed with PBS. For indirect IF, the sections were incubated with anti–mouse or –rabbit IgG conjugated with fluorescein isothiocyanate, tetramethyl rhodamine thiocarbamoyl, or sulfonated 7-amino-4-methyl coumarin-3 acetic acid. Colocalization studies were preformed by direct IF; anti–Kv1.4, 4.2, and 4.3 antibodies were directly conjugated with the above fluorochromes. Antimyosin antibody was used as a positive control. Sections incubated (a) without primary antibodies or (b) preabsorbed primary antibodies (anti– Kv1.4, 4.2, and 4.3 antibodies previously incubated with myocytes) were used as negative controls. To demonstrate the localization patterns within isolated myocytes, cells were stained with DiIC 18 (dioctadecyl 3,3,3′,3′ tetramethyl indocarbo cyanine) and propidium iodide markers that label the sarcolemma and nucleus, respectively. The antibody binding and staining patterns in isolated myocytes were analyzed by taking 1 μm optical Z sections through the cells. Fluorescence was detected using an inverted confocal microscope equipped with ArKr, HeNe, and HeCd laser beams under appropriate excitation and emission wavelengths for each fluorochrome. For numerous reasons (i.e., monoclonal [Kv1.4] versus polyclonal [Kv4.2, Kv4.3] antibodies employed, possible differences in number, accessibility, and affinity of targeted epitopes, etc.), a direct quantitative comparison of fluorescence intensities between each of the individual antibodies employed (to estimate and/or compare absolute Kv1.4, Kv4.2, and Kv4.3 α subunit protein expression levels) was precluded. We therefore wish to emphasize that the specific immunoreactivities/fluorescent intensities that were obtained for any one specific α subunit cannot be quantitatively compared with those obtained for another α subunit type; only qualitative and/or relative comparisons of individual α subunit protein levels are valid. IF profiles for individual proteins within transverse sections across the ventricular wall were analyzed by confocal microscopy, and for comparative purposes the relative fluorescent intensity values of each α subunit protein were normalized to the maximum fluorescence obtained for that α subunit (i.e., maximum relative intensity = 100%). The IF results presented on enzymatically isolated LV epi versus LV endo myocyte preparations measure Kv1.4, Kv4.2, and Kv4.3 α subunit protein expression levels within specific myocyte types. As will be described, these measurements indicate marked heterogeneous expression levels of these three α subunits between LV epi versus LV endo myocytes. These heterogeneous myocyte α subunit expression levels will contribute to net IF patterns obtained from intact ventricular tissue sections. However, in ventricular tissue Kv1.4, Kv4.2, and/or Kv4.3 α subunits may be expressed not only in myocytes but also in numerous nonmyocyte cell types, including fibroblasts, endothelial cells, smooth muscle cells, and neurons. To date, we have not analyzed the distribution of candidate I to α subunit expression levels in these various nonmyocyte cell types. We therefore wish to emphasize that the IF patterns obtained from sagittal sections of ventricular tissue cannot at present be attributed exclusively to Kv1.4, Kv4.2, and/or Kv4.3 α subunit protein expression within myocytes. Under our recording conditions (Na + - and Ca 2+ -free saline, 500 μM CdCl 2 ; see methods ) in both ferret LV epi and LV endo myocytes, calcium-independent transient outward potassium currents could be routinely recorded in response to depolarizing voltage clamp steps. Representative examples of these transient outward currents in both LV epi and LV endo myocytes in response to either 500- (LV epi) or 1,000-ms (LV endo) depolarizing clamp pulses applied from a holding potential (HP) of −70 mV are illustrated in Fig. 1 , A and B. On initial inspection, the transient outward currents in both myocyte types displayed somewhat similar basic macroscopic gating characteristics: both activated very rapidly (within milliseconds, with activation of the peak LV endo current being generally faster, resulting in its peak being frequently obscured due to overlap with the capacitive transient), and then displayed a slower phase of macroscopic inactivation to a final steady state level (i.e., a noninactivated sustained component). Additional measurements (data not shown) wherein the [K + ] o was varied (1–100 mM) indicated that the amplitude of the transient components of these two currents was indeed a function of [K + ] o , verifying that they were K + currents. Following the convention established in previous studies , we defined the inactivating transient component (i.e., I peak − I sustained ) of these currents as “I to .” We will refer to these two calcium-independent I to phenotypes in ferret LV epi and LV endo myocytes as I to,epi and I to,endo , respectively. There were no significant differences between LV epi versus LV endo myocytes in either (a) resting membrane potential (measured in control solution before establishing voltage clamp; LV epi, V m = −72.5 ± 0.7 mV, n = 9; LV endo, −72.6 ± 0.6 mV, n = 10) or (b) density of the inwardly rectifying K + current, I K1 (measured in response to 500-ms hyperpolarizing voltage clamp pulses applied to −80 to −120 mV). However, as illustrated in Fig. 1 , C1 and C2, there were significant differences in both activation thresholds and mean peak current–voltage relationships between I to,epi (defined as I peak − I 500 ms , n = 7) and I to,endo . I to,epi activated at −10 to 0 mV and had a peak density at +20 mV of +4.03 ± 0.52 pA/pF. In contrast, I to,endo activated at −30 to −20 mV and had a much lower peak current density at +20 mV of +0.77 ± 0.16 pA/pF. To further quantify activation characteristics, attempts were made to measure steady state activation relationships using a conventional saturating tail current protocol . This protocol could be successfully applied to LV I to,epi , giving a mean activation curve that could be well described as a single Boltzmann relationship (V 1/2 = +22.5 mV, slope factor [ k ] = 7.95 mV, n = 5; data not shown). Unfortunately, the deactivation kinetics of I to,endo tail currents were too rapid and the tails too small (i.e., obscured by the capacitive transient) to allow for resolution and construction of an activation relationship. The mean steady state inactivation relationships for I to,epi ( n = 7) and I to,endo ( n = 8) are illustrated in Fig. 2 A (protocols illustrated in inset). Steady state inactivation relationships for both I to phenotypes could be well described as single Boltzmann relationships. However, there were significant differences among the two inactivation relationships in both V 1/2 and k values (I to,epi : V 1/2 = −9 mV, k = 5.45 mV; I to,endo : V 1/2 = −34 mV, k = 8.05 mV). To determine the kinetics of macroscopic inactivation, the declining phases of the two LV I to phenotypes were analyzed. Inactivation of I to,epi was well described as a single exponential , with a mean time constant at +50 mV of 75.7 ± 4.8 ms ( n = 9). Inactivation time constants became essentially independent of membrane potential depolarized above +20 mV . In contrast, the kinetics of inactivation of I to,endo at potentials depolarized above +30 mV required two exponentials for an adequate fit , with mean time constants at +50 mV of τ 1 = 80.3 ± 9.7 ms and τ 2 = 466.4 ± 128.5 ms (ratio of relative initial amplitudes A 1 /[A 1 + A 2 ] = 0.653 ± 0.075). However, in the less depolarized range of potentials (−10 to +30 mV), it was difficult or impossible to obtain reliable double exponential fits , thereby precluding a more quantitative analysis. The kinetics of recovery from inactivation of the two LV I to phenotypes were determined at a HP of −70 mV using a conventional double pulse protocol . In the majority of LV epi myocytes ( n = 7/9, 78%), recovery was rapid and could be well described as a single exponential with a mean τ rec = 51.2 ± 2.8 ms . In two additional LV epi myocytes, there was a major fast component of recovery (accounting for 90 and 95% recovery within 200–250 ms), followed by a slower phase (on the order of hundreds of milliseconds), with recovery being complete within 2–3 s (data not illustrated). In contrast, in the majority of LV endo myocytes ( n = 8/11, 73%), recovery was quite slow and could be reasonably described as a single exponential with a mean τ rec = 3,001.8 ± 447.1 ms . However, in three LV endo myocytes, recovery was rapid (single exponential, τ rec = 66.7 ± 3.4 ms; data not shown); i.e., comparable to I to,epi . In summary, while in the majority of LV epi and LV endo myocytes studied there was a clear distinction between “pure” rapid recovery (I to,epi ) versus “very slow” recovery (I to,endo ), this kinetic differentiation was not absolute. However, in no instance was an exclusively “very slow recovery” pattern ever observed in an LV epi myocyte. Due to these marked differences in recovery kinetics, it would be predicted that cumulative inactivation would be a prominent characteristic of I to,endo but not I to,epi . As predicted, during rapid and repetitive voltage-clamp pulse trains applied to +50 mV the slowly recovering I to,endo phenotype displayed marked cumulative inactivation while the I to,epi phenotype did not. Heteropoda toxins (HPTXs) are peptides (30–33 amino acids) isolated from the venom of the spider Heteropoda venatoria . HPTXs block both native I to channels in rat ventricular myocytes and Kv4.2 channels expressed in Xenopus oocytes; in contrast, HPTXs have no significant effects on expressed Kv1.4 channels . To determine whether we could pharmacologically distinguish between ferret LV I to,epi and I to,endo , we conducted a preliminary analysis of the effects of HPTX2 . 150 nM HPTX2 rapidly and reversibly blocked I to,epi without any significant effects on the sustained current remaining at 500 ms . However, the degree of block was potential dependent, with block being relieved with progressive depolarization. Block decreased from 75.6 ± 5.3% at +10 mV to 21.2 ± 4.8% at +70 mV . Assuming a simple single site binding model, the estimated apparent K d for HPTX2 block ranged from 105 nM at +20 mV to 559 nM at +70 mV . In contrast, 150 nM HPTX2, when perfused over a period of 5–7 min, failed to produce any significant block of the major slowly recovering I to,endo phenotype elicited at +50 mV . A comparative summary of the gating characteristics of the two major I to phenotypes in ferret LV epi and LV endo myocytes is presented in Table I . This table also summarizes the characteristics of the rapidly recovering I to phenotype in ferret RV myocytes . While there are quantitative differences in specific gating characteristics among RV and LV epi I to phenotypes (e.g., τs of inactivation, recovery), overall these two current systems are very similar. The rapidly inactivating Kv1.4, Kv4.2, and Kv4.3 clones have all been recently proposed to form the molecular basis of the cardiac myocyte calcium-independent I to . Therefore, the FISH and IF measurements to be described in the next section were designed to determine whether there was a differential distribution of Kv1.4, Kv4.2, and/or Kv4.3 α subunits between ferret LV epi and LV endo myocytes, and whether such a differential distribution correlated with the two major functional I to phenotypes described in the preceding sections. To determine the specificity of the antibodies employed, IF and immunoblot analyses were first performed. IF experiments were performed on Xenopus oocytes injected with K + channel mRNA or sham-injected with water . Oocytes injected with Kv4.2 mRNA ( n = 7) only reacted with the Kv4.2 antibody , while oocytes injected with the short form of Kv4.3 mRNA ( n = 10) only reacted with the Kv4.3 antibody (G–I). Neither antibody stained the sham-injected oocytes . Immunoblot analyses for each antibody were then performed on four different protein preparations obtained from ferret LV epicardial and endocardial regions: (a) sarcolemmal membrane enriched fractions , (b) cytosolic-enriched fractions (lanes 4 and 5), (c) particulate-enriched fractions (lanes 6 and 7), and (d) whole-tissue homogenates (lanes 8 and 9). Among the sarcolemmal membrane–enriched fractions, prominent and specific antibody binding patterns were obtained. The Kv1.4 antibody recognized a single specific band at ∼95–100 kD in the LV endo fraction ; in contrast, there was virtually no reactivity of the Kv1.4 antibody in the LV epi fraction (lane 2). The Kv4.2 antibody specifically recognized a single prominent band at ∼75 kD in the LV epi fraction and a similar but weaker band in the LV endo fraction (lane 3). Finally, the Kv4.3 antibody recognized a single band at ∼75 kD in both LV epi and LV endo fractions . In combination with earlier results , our IF and immunoblot data strongly suggest that the three antibodies employed detect and specifically bind to Kv1.4, Kv4.2, and Kv4.3 α subunits expressed in ferret LV epi and LV endo tissues. These results also suggest that the majority of α subunit proteins detected by the antibodies are located at cell surfaces. To determine specific antibody binding patterns to isolated LV epi and LV endo myocytes, confocal microscopy was employed. Binding patterns for each of the three antibodies were determined by measuring fluorescence intensity profiles in successive optical z sections (1-μm thickness) taken through the entire width of each myocyte type. Fig. 6 illustrates a representative series of such optical z sections (every third section is illustrated) measured from isolated LV epi (anti–Kv4.2, C1–C6; anti–Kv4.3, D1– D6) and LV endo (anti–Kv1.4, E1–E6) myocytes. For controls, standard membrane markers for the sarcolemma and nucleus (propidium iodide; B1–B6) were used. The fluorescence patterns obtained in the successive optical z sections indicated that binding of all three antibodies was localized to the outer regions of myocytes, thereby suggesting localization to sarcolemmal proteins. In combination with our immunoblot data , these z section results strongly suggest that sarcolemmally associated Kv1.4, Kv4.2, and/or Kv4.3 α subunits could function as the molecular substrates underlying the two different functional I to phenotypes in ferret LV epi and LV endo myocytes. To gain further insights into the distribution and identification of the specific α subunits underlying the I to,epi and I to,endo phenotypes, measurements of both (a) mRNA transcripts and (b) expressed protein using the Kv1.4, 4.2, and 4.3 α subunit–specific antibodies were conducted in parallel on samples of enzymatically isolated LV epi and LV endo myocytes. The results of these measurements are summarized in Table II (mean results from isolated myocyte samples obtained from a total of seven ferret left ventricles; number of myocytes analyzed per sample per anatomical region, 500). Messenger RNA (FISH) for Kv1.4 was found in the majority of LV epi and LV endo myocytes at a comparable percentage, while mRNA transcript for both Kv4.2 and 4.3 was more abundantly expressed in LV epi than LV endo myocytes. When the percentage of myocytes exhibiting Kv α subunit proteins was analyzed using IF, a different pattern emerged: (a) Kv1.4 protein was expressed in the majority of LV endo myocytes (57.9 ± 2.8%), but was essentially absent in LV epi myocytes; and (b) Kv4.2 and 4.3 were each more predominantly expressed in LV epi (56.8 ± 1.7% and 45.3 ± 1.8%, respectively) than LV endo (21.2 ± 2.4% and 34.6 ± 1.3%, respectively) myocytes. Similarly, when α subunit protein colocalization was analyzed: (a) Kv1.4 + Kv4.2 colocalized more in LV endo (10.3 ± 1.7%) versus LV epi (1.8 ± 1.1%) myocytes, (b) Kv1.4 + Kv4.3 colocalization was small to virtually absent in both myocyte types, and (c) Kv4.2 + Kv4.3 colocalized mainly in LV epi (23.6 ± 0.6%) versus LV endo (12.1 ± 1.2%) myocytes. In summary, our results indicate that both Kv4.2 and Kv4.3 α subunits are expressed in ferret LV epi and LV endo myocytes, with expression of both being relatively more abundant in LV epi myocytes. In contrast, the Kv1.4 α subunit is abundantly expressed only in LV endo myocytes. Furthermore, while the mRNA transcript levels of both Kv4.2 and Kv4.3 in isolated myocytes correlated in general with the levels of expressed α subunit protein, such a correlation did not exist for Kv1.4. In this final section, we extend the parallel FISH and IF measurements described above for isolated LV myocytes to sagittal sections of whole intact ventricular tissue. These measurements were conducted so as to gain initial insights into overall ventricular tissue distribution patterns of Kv1.4, Kv4.2, and Kv4.3 mRNA transcript and expressed α subunit proteins. Representative parallel FISH and IF results obtained from such ventricular tissue sections are illustrated in Fig. 7 . As controls, sagittal sections taken from the ferret ventricle were subjected to hybridization with TnIC antisense or sense probes . These data demonstrated that the entire sections were competent for FISH assays, and that these assays produced very low background when using the sense probe for TnIC. Similar results were obtained with sense probes derived from Kv1.4, Kv4.2, and Kv4.3 (data not shown). IF obtained using antimyosin antibody also gave results similar to those illustrated in Fig. 7 B (data not shown), demonstrating that these sections were also competent for IF assays. From these measurements, it was concluded that the observed localization patterns were not artifacts of the tissue preparation. Finally, as controls for the indirect IF results, assays conducted with secondary antibodies (fluorochrome conjugated anti–mouse or –rabbit IgG) alone demonstrated that our indirect fluorescent signals were due to specific binding to the primary antibodies . Using FISH, Kv1.4 mRNA was abundantly and uniformly expressed throughout all regions of the ferret ventricle . In contrast, Kv4.2 mRNA was heterogeneously expressed, being prominent in apex, RV, LV epi (from both apex to base), and both basal LV endo and septum, while it was markedly reduced to absent in both apical LV endo and septum . Kv4.3 mRNA was more uniformly expressed, although it was relatively more abundant in the apex and apical LV epi and was reduced in the RV . Similar mRNA distribution results for all three α subunits were observed in sagittal sections obtained from a total of seven ferret hearts. In contrast to the FISH results, indirect IF analysis indicated that Kv1.4 α subunit protein was essentially absent from the RV, the LV epi, and the basal region of the LV, but was uniformly expressed in the apical LV endo and midmyocardium (free wall and septum) . Kv4.2 α subunit protein was expressed abundantly in the apex, RV, and LV epi (apex and base) and the basal LV endo and septum, but was markedly reduced to absent in the apical LV endo . Finally, Kv4.3 α subunit protein was more uniformly expressed, although it was relatively low to absent in RV and relatively more abundant in the apex and LV epi compared with LV endo . Therefore, similar to the isolated myocyte studies, results from the ventricular sections indicate that there was a general correspondence between differential Kv4.2 and 4.3 mRNA transcript levels and expressed α subunit proteins, while such a correspondence did not hold for Kv1.4. A further demonstration of the heterogeneity of the three α subunits across the ventricular wall is shown by the relative fluorescence intensity profiles measured transversely at the indicated basal (F–J, 3), mid-ventricular (F–J, 4) and apical (F–J, 5) regions. Representative sagittal section colocalization data for Kv1.4, Kv4.2, and Kv4.3 α subunits obtained using direct IF are illustrated in Fig. 8 for Kv1.4 + Kv4.2 (A: Kv1.4, red; Kv4.2, green), Kv1.4 + Kv4.3 (B: Kv1.4, red; Kv4.3 green), and Kv4.2 + Kv4.3 (C: Kv4.2, green; Kv4.3, red; note that in these panels colocalization is represented by a yellow to orange color; refer to legend for details). Fig. 8 D illustrates the colocalization pattern of all three α subunit proteins (Kv1.4, blue; Kv4.2, green; Kv4.3, red). From Fig. 8 D it can be seen that there was a relative abundance of Kv4.2 (green) and Kv4.3 (red) in the epicardial regions and Kv1.4 (blue) in the apical endocardial region of the LV, clearly establishing a regional localization. There was also some colocalization of Kv4.2 + Kv4.3 (yellow to orange) in the epicardial regions and Kv1.4 + Kv4.3 (purple to pink) in the endocardial region of the LV and the septum. In summary, α subunit protein expression distribution patterns similar to those illustrated in Figs. 7 and 8 were obtained from at least three ferret hearts (Kv4.3, n = 3; Kv1.4 and Kv4.2, n = 6). The general distribution patterns of the three α subunits were as follows: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endo, and approximate inner 75% of the LV free wall; (b) Kv4.2 was localized primarily to RV free wall, epicardial layers of the LV, and the base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV and base of the heart. Therefore, in the intact ventricle, there is a substantial heterogeneous distribution of I to α subunits not only from epicardium to endocardium but also from apex to base. Two distinct I to phenotypes, which we have designated I to,epi and I to,endo , are differentially expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes. Under our recording conditions (Na + - and Ca 2+ -free saline, 500 μM Cd 2+ ) I to,epi and I to,endo display significant differences in activation, inactivation, and recovery characteristics and sensitivity to block by HPTX2. In combination, these results strongly suggest that I to,epi and I to,endo are produced by (at least) two distinct potassium channel α subunits that have different molecular mechanisms governing inactivation and recovery characteristics . In addition to different underlying α subunits, other (presently uncharacterized) factors may also be contributing to these kinetic differences, including differences in associated β subunits and homomeric versus heteromeric α subunit assembly . Possibly the most functionally significant difference between I to,epi and I to,endo resides in their kinetics of recovery from inactivation. In the majority of LV epi and LV endo myocytes studied recovery at a HP of −70 mV was well-described as a single exponential process; however, the mean τ rec of I to,endo was ∼60× slower than I to,epi . As a result, I to,endo displays marked cumulative inactivation, while I to,epi does not. These two major I to phenotypes will therefore exert significantly different frequency-dependent modulatory effects on action potential morphology and repolarization characteristics. For example: (a) in LV epi myocytes, the predicted effects of reducing I to,epi should only become prominent at high heart rates; and (b) in LV endo myocytes, the modulatory effects of I to,endo should be minimal at normal heart rates (due to cumulative inactivation) but could become significant at low rates. In addition, if metabolic factors (e.g., myocyte redox status, second messengers, phosphorylation) are capable of modulating I to,endo inactivation/recovery characteristics , then under certain conditions I to,endo could become a significant repolarizing current at normal heart rates. In preliminary action potential (AP) measurements, we have obtained results consistent with these general functional predictions in some ferret LV epi and LV endo myocytes. However, as would be predicted by our IF results, we have also observed significant variability in both AP morphologies and frequency-dependent characteristics (0.2–1 Hz), particularly among LV endo myocytes (D.L. Campbell, unpublished observations). It is therefore important to note that our previous FISH and IF measurements demonstrated marked heterogeneous expression patterns of multiple potassium channel α subunit mRNA transcripts and proteins within different anatomical regions and myocytes of the ferret heart. In combination with this previous data, our preliminary AP recordings suggest that, in addition to I to,epi and I to,endo , other repolarizing currents are also heterogeneously expressed. In conjunction with future current clamp studies on AP morphologies, the kinetic properties of each of these different repolarizing currents need to be quantitatively analyzed before a more realistic understanding of LV epi versus LV endo myocyte repolarization can be achieved. Our immunolocalization data on isolated LV epi and LV endo myocytes clearly indicate a correlation between the expression patterns of Kv1.4, 4.2, and 4.3 α subunits and the two major functional LV myocyte I to phenotypes. Specifically, our data strongly suggest that Kv4.2/4.3 α subunits largely underlie I to,epi , and Kv1.4 α subunits largely underlie I to,endo . This hypothesis is further strengthened by the fact that both I to,epi and expressed Kv4.2/4.3 α subunits display rapid (although not identical) recovery kinetics, while both I to,endo and expressed Kv1.4 α subunits display very slow (although not identical) recovery kinetics with marked cumulative inactivation. While exclusively rapid versus exclusively slow recovery patterns are the dominant phenotypes in ferret LV epi versus LV endo myocytes, there was a subpopulation of both (a) LV endo myocytes (27%; n = 3/11) that displayed rapid single exponential recovery kinetics and (b) LV epi myocytes (22%; n = 2/9) that displayed an additional slower phase of recovery (on the order of hundreds of milliseconds). The percentage of LV endo myocytes displaying rapid recovery was therefore similar to the sum of the percentage of isolated LV endo myocytes displaying colocalization of Kv1.4 + Kv4.2 (10.3 ± 1.7%) and Kv1.4 + Kv4.3 (6.4 ± 2.3%) α subunits. In contrast, the percentage of LV epi myocytes displaying a slower component of recovery was not comparable to the percentage of isolated LV epi myocytes displaying colocalization of Kv1.4 + Kv 4.2 and Kv1.4 + Kv4.3 α subunits. Although caution must be exercised in directly comparing these two sets of data, the similarity does allow for speculation that the coexpression of Kv1.4 and Kv4.2 and/or Kv4.3 α subunits within the same LV endo myocyte may underlie the subpopulation of rapidly recovering I to,endo phenotypes. At present, our data cannot account for the subpopulation of LV epi myocytes that displayed an additional slow component of recovery. However, in no instance was a “purely slowly recovering” I to phenotype observed in an LV epi myocyte. This correlates very well with the fact that Kv1.4 protein was virtually absent in isolated LV epi myocytes. Finally, while to date we have not specifically analyzed I to α subunit expression in ferret isolated RV myocytes, our IF results on intact ventricular sections (see Interpretive Limitations in methods ) have implications for our previous study of the I to phenotype in ferret isolated RV myocytes . Ferret RV myocytes almost exclusively express a uniform I to phenotype with rapid recovery kinetics. The RV I to phenotype would therefore appear to correlate with the virtual absence of Kv1.4 and the uniformly abundant expression of Kv4.2 α subunits in ferret intact RV tissue. Furthermore, the RV I to phenotype displays marked closed state reverse-use–dependent block by 4-aminopyridine , a property shared by expressed Kv4.2 channels but not by Kv1.4 channels . Sanguinetti et al. have reported differential block of expressed Kv1.4 and Kv4.2 channels by HPTXs (Kv1.4, insensitive; Kv4.2, sensitive). These investigators observed that HPTX block of Kv4.2 was relieved with progressive depolarization, while at a fixed potential the dose–response curve could be well-described using a Hill equation formulation with voltage-dependent coefficients ranging from one to two. The effects of HPTXs on Kv4.3 were not determined in this study. However, given the extensive sequence homology in the core region between Kv4.2 and Kv4.3, it is likely that HPTXs would have similar effects on these two α subunits. The results of Sanguinetti et al. , in combination with our HPTX2 results (I to,epi , sensitive; I to,endo , insensitive), further strengthens the hypothesis that in ferret LV endo and LV epi myocytes Kv1.4 and Kv4.2/4.3 α subunits, respectively, are the major substrates underlying I to,endo and I to,epi . The characteristics of the two major I to phenotypes that we measured in ferret LV epi and LV endo myocytes are very similar to those recently reported for the two major I to phenotypes in human left ventricular epicardial and endocardial myocytes , although there are quantitative differences. In both ferret and human LV, the major I to,epi phenotype is expressed at relatively high current densities, activates and inactivates at relatively depolarized potentials, and recovers rapidly (and hence displays little to no cumulative inactivation). Similarly, in both ferret and human LV, the major I to,endo phenotype is expressed at relatively low current densities, activates and inactivates at more hyperpolarized potentials, and recovers slowly (and hence displays cumulative inactivation). This raises the possibility that there may be a differential distribution of Kv1.4, 4.2, and/or 4.3 α subunits in human LV epi and LV endo myocytes similar to that which we observe in ferret. In contrast, I to phenotypes of both ferret and human LV myocytes appear to differ from those of rabbit and canine ventricular myocytes. In rabbit ventricle, only one major slowly recovering I to phenotype appears to be dominant but expressed at different densities in epicardial versus endocardial myocytes . Hence, in rabbit, Kv1.4 may be the dominant α subunit expressed in both epicardium and endocardium. In canine LV, there also appears to be one major expressed I to phenotype displaying relatively slow recovery kinetics but different levels of expression in LV epi, LV midmyocardial, and LV endo myocytes . However, it has recently been proposed that Kv4.3 α subunits underlie the canine LV I to . Our results would therefore suggest, at least with regard to the role(s) of multiple and distinct I to phenotypes in modulation of LV repolarization, that the ferret may provide a better comparative cellular model for the human heart than other more commonly studied species. To conclude, we wish to point out that our parallel FISH and IF results on both isolated LV myocytes and intact ventricular tissue sections yield additional important insights into presently unrecognized but potentially serious limitations of these techniques as they are now commonly employed. Three potentially very important, and interrelated, implications of our results follow. For both Kv4.2 and 4.3, there was a general concordance between the distribution of transcript and α subunit protein in the ferret ventricle. In contrast, for Kv1.4, there was a marked disparity between the distribution of transcript and α subunit protein expression, possibly suggesting the presence of some factor that affects translation of the α subunit; e.g., an ancillary β subunit may be needed for protein assembly and insertion into the sarcolemma . These very interesting results suggest differences in transcriptional regulation of the different I to α subunit proteins throughout the heart. However, they also clearly indicate that conclusions on the presence of candidate ion channels and/or α subunits within specific individual myocyte types cannot always be reliably made based solely on measurements of transcript mRNA levels obtained from whole tissue and/or whole heart preparations. Our results suggest that application of specific IF techniques allow for a more accurate analysis of expressed α subunit proteins within specific anatomical regions, important data that can then be directly correlated with functional patch clamp measurements on myocytes isolated from the same specific regions. Our present data corroborates our previous in situ hybridization results on Kv1.4, 4.2, and 4.3 in isolated myocytes prepared from different anatomical regions of the ferret heart . Our new data extend these previous observations by examining, in adjacent sagittal sections, the distribution of several corresponding expressed α subunit proteins. Our present results indicate that, due to significant heterogeneous expression of I to phenotypes and corresponding α subunits throughout distinct regions of the ventricle , very careful attention must be paid to the exact anatomical region from which tissue and/or isolated myocyte samples are taken for analysis. While analysis of protein distribution within individual myocyte types is important for the ultimate identification of the molecular substrates underlying currents within that particular myocyte type, analysis of the distribution of Kv α subunit proteins in tissue sections provides important information concerning the distribution of the different Kv α subunits throughout the ventricular wall. Such information is important since in the intact heart the net process of repolarization results from the overall distribution patterns of multiple potassium channel α subunit proteins expressed throughout the entire ventricular myocardium. However, as discussed (see Interpretive Limitations in methods ), whole-tissue IF analysis suffers from the complications of potential α subunit protein expression in various nonmyocyte cell types. For this reason, we can not at present attribute the fluorescence signals obtained from the intact ventricular sagittal sections exclusively to I to α subunit protein expression in myocytes. Nonetheless, our IF results on isolated LV epi versus LV endo myocytes clearly demonstrate heterogeneous expression gradients of Kv1.4, 4.2, and 4.3 α subunits that essentially parallel those observed across the intact LV wall. These results indicate that heterogeneous myocyte α subunit expression gradients are significantly contributing to the net LV epicardial-to-endocardial fluorescence signals measured in the sagittal tissue sections. Our sagittal section IF results would therefore suggest that the distribution of multiple I to phenotypes in the mammalian heart, and their potential effects upon net repolarization characteristics, may be significantly more complicated than presently suspected. In conclusion, our results demonstrate (a) the presence of (at least) two major functionally distinct I to phenotypes in ferret LV epi and LV endo myocytes, (b) distinct heterogeneous expression patterns (both epicardial-to-endocardial and apical-to-basal) of Kv1.4, 4.2, and 4.3 α subunits in ferret intact LV tissue, and (c) a parallel α subunit expression gradient at the level of isolated LV epi and LV endo myocytes, thereby strongly suggesting that Kv4.2/4.3 α subunits largely underlie I to,epi , and Kv1.4 α subunits largely underlie I to,endo . | Study | biomedical | en | 0.999998 |
10189364 | Fibroblasts of the diploid MRC-5 line (a gift of Dr. J. Willey, Medical College of Ohio) and the diploid CCD-34Lu cell line (American Type Culture Collection), both derived from human lung tissue, were grown as monolayers directly on glass slides in RPMI 1640 or EMEM containing l -glutamine ( GIBCO BRL ), 10% FBS ( GIBCO BRL ), penicillin, gentamicin, and sodium bicarbonate ( Amersham Life Sciences ), respectively. The cells were fixed in situ with Carnoy's solution just before confluence. Human lymphocytes were grown in RPMI 1640 with the addition of phytohemagglutinin ( Amersham ) for 72 h, fixed in Carnoy's, and dropped onto glass slides from 10 cm. The slides were not flamed, but were allowed to air-dry and were stored until hybridization. In some experiments, CCD-34Lu cells were fixed in 4% paraformaldehyde in PBS and stored without drying in 95% alcohol at −20°C until hybridization . Centromere-specific probes, directly labeled with FluorX (green fluorescence) or Cy3 (red-orange fluorescence), were used when available ( Amersham ). For the remaining chromosomes, chromosome “paints,” labeled with Spectrum orange or Spectrum green, were used (Vysis), and the brightest point on each “paint image” was used as the location of the centromere. For FISH, slides were incubated in a 2× SSC solution (pH 7.0) for 30 min, followed by dehydration. The centromeric probe mixtures consisted of 2 μl of Cy3-labeled centromeric probe, 2 μl of FluorX-labeled centromeric probe, and 10 μl of hybridization solution (50% formamide/ 2× SSC/10% dextran sulfate). The centromeric-paint probe mixtures consisted of 1 μl of Spectrum orange or green paint probe, 2 μl of FluorX or Cy3 centromeric probe, 1 μl of ddH 2 O, and 7 μl of hybridization solution. The probe mixtures were denatured at 70°C for 5 min and placed at 4°C until use. Cells hybridized to the centromeric and the paint-centromeric probe mixtures were denatured for 2 and 5 min, respectively, in 70% formamide/2× SSC solution at pH 7.0. The slides were incubated overnight with probe solution in a humidified chamber at 43°C. The slides incubated with the paint-centromeric and the centromeric probes were washed in 50–65% formamide/2× SSC solution (pH 7.0), 2× SSC, and 2× SSC with NP-40 or PBD (pH 8.0), respectively, and counterstained with DAPI. The appropriate number of centromeres were always clearly localized in the Carnoy-fixed mitotic and interphase cells . The paraformaldehyde-fixed CCD-34Lu cells gave relatively dim probe localization under a variety of denaturation times (2–6 min) when compared with the Carnoy-fixed cells. However, treatment of the paraformaldehyde-fixed cells with a weak solution of HCl (200 mM in PBS) for 20 min at room temperature before a 3-min denaturation allowed detection of the appropriate number of fluorescence signals in the majority of rosettes. The Cy3 and Spectrum orange fluorochromes were localized with a rhodamine-specific filter cube, BP510-560, FT580, LP590, in a Zeiss microscope under epifluorescence optics with a Neofluar 100× oil immersion lens (NA 1.30; Carl Zeiss, Inc. ). The FluorX and Spectrum green fluorochromes were visualized with filter cube BP450-490, FT510, LP520, and a G365, FT395, LP420 filter cube was used for the DAPI stain. Analogue images from a CCD camera mounted on the microscope were digitized and processed for removal of extraneous background fluorescence by Probevision software (Applied Imaging Corp. [AI]). The early and mid-anaphase mitotic rings are perpendicular to the slide surface, and FISH-localized chromosomes in these cells were often in slightly different focal planes. When this occurred, the objective was set at an intermediate focal plane between the two probes, which appeared as slightly larger and less bright spots of light than perfectly focused probes. AI image analysis transforms were used to select the brightest points in each of the defocused spots as the location of probe fluorescence. The AI fluorescence microscopy system separately acquires three black and white images at the emission wavelength of the fluorochrome being localized. The black and white images are combined into one pseudocolor image without any movement or alignment changes. Each image was converted into a color graphic overlay (AI) and further processed with Adobe Photoshop (Adobe Systems Inc.) and Probe Ratio software (JVB Imaging). Data were stored and analyzed with the Quatro Pro spreadsheet (Borland) and the SPSS statistical programs (SPSS Inc.). The emitted light from the contrasting fluorochromes has different refractive indices in the microscope objective. To test whether the varying focal planes and emission spectra caused significant shifts in image positions, we hybridized female lymphocytes with the FluorX paint probe and the Cy3 centromeric probe for the X chromosome. The two probes showed a perfect positional correspondence for all cells measured , ruling out significant spectral aberrations and alignment problems. After S-phase, the newly replicated sister chromatids condense in prophase , and many, if not all, prophase cells form a tight ring of chromosomes parallel to the slide surface called the prometaphase rosette . The prometaphase rosettes progress directly to less compact metaphases , followed shortly by anaphase . The early and mid- anaphase mitotic rings are perpendicular to the slide surface. We found, similar to Nagele et al. , that it was difficult to determine the positions of FISH-localized chromosomes in metaphase figures, which often have partially broken or folded mitotic rings . This was not the case for the more compact rosettes and anaphases . The symmetry of chromosomal positions in >99% of the daughter early and mid-anaphases established that the relative chromosomal positions in the living early and mid-anaphases were maintained after fixation. The mitotic rings of late-anaphases were often parallel to the slide surface . The proportion of prometaphases forming flat rosette rings was graded in consecutive lymphocytes and MRC-5 cells. Because it had been reported that only “perfect” rosettes were suitable for analysis of chromosomal positions , MRC-5 and lymphocyte rosettes were further classified as being perfect (compact, even, and unbroken mitotic rings), “slightly spread” (slight separation of some chromosomes and/or some central asymmetry), or “gap” (<10% broken area in the ring) rosettes. Perfect rosettes were found for 38% (101/261) and 9% (48/551) of the lymphocyte and MRC-5 prometaphases, respectively. However, no differences in the angular separations in perfect, slightly spread, or gap rosettes were found for any of the cell lines (data not shown), and all three rosette types were subsequently measured, giving estimated sampling frequencies of 90% (234/261) and 29% (162/557) of the lymphocyte and MRC-5 prometaphases, respectively. Fig. 1 D shows a prometaphase or late-anaphase mitotic ring parallel to the slide surface with the two homologues of chromosome 17 separated by 180°. A change of position of one homologue leads to two separation angles between these chromosomes, one <180° and one >180°. The lower angle was measured, allowing a 0–180° separation range between two rosette chromosomes. However, it was necessary arbitrarily to select a center point to place a measuring grid over the ring . To test the reproducibility of this step, we performed two sets of measurements of the same prometaphase rosettes, with the second measurement set performed without knowledge of the prior location of each rosette's center point . In Fig. 1 E, the ratios of the first to second angular measurements for each rosette are plotted on the y-axis against the mean value of the two measurements on the x-axis. There was considerable variability between the two measurement sets, especially for measurements of smaller angular separations . The variability in our study seemed random, because the ratios were both above and below the value of one . Consecutive, widely separated CCD-34Lu and MRC-5 chromosomal masses were graded as being late-anaphases , telophases , or of indeterminate morphology (not shown), leading to the following classifications: both chromosomal masses being late-anaphases (CCD-34Lu, n = 18 pairs; MRC-5, n = 18 pairs); both being telophases (CCD-34Lu, n = 14 pairs; MRC-5, n = 30 pairs); and being of mixed/indeterminate morphology (CCD-34Lu, n = 40 pairs; MRC-5, n = 22 pairs). The angular separations in nonpaired, i.e., individual, coded images of these chromosomal masses were measured one at a time, using the geometric centers of each chromosomal mass to center the measuring grid . The angular chromosomal separations cannot be measured directly in early and mid-anaphase mitotic rings, which are perpendicular to the slide surface . Linear distances were measured between the anaphase chromosomes and then analyzed to gain an estimate of the native chromosome sequence as detailed in the Appendix . The angular separations measured between the homologues of chromosomes 11 ( n = 103) and 17 ( n = 203) in MRC-5 rosettes, chromosome 17 in male lymphocyte rosettes ( n = 100), chromosome 7 in female lymphocyte rosettes ( n = 104), and chromosomes X and 7 in the CCD-34Lu rosettes ( n = 156) were highly variable . No evidence was found for fixed ranges of separation between these homologues on the mitotic ring, as equal numbers of homologues were separated by <90° and by >90° . If the chromosomes are in fixed positions in male cells, the angular separations between the X chromosome and the same two somatic homologues should be identical for every rosette . This was not the case for measurements of the X and 17 chromosome homologues made on male MRC-5 and lymphocyte rosettes, where widely variable angles of separation were found . Random separations of homologous rosette chromosomes were also found for all of the individual lymphocyte chromosomes, MRC-5 chromosomes 11 and 17, and CCD-34Lu chromosomes X and 7 (see Appendix , Table I ). Furthermore, the distributions of the nearest angular separations between the somatic chromosomes to either the X or Y chromosome in male lymphocytes rosettes were also highly variable (see Appendix , Table II ). We measured the relative chromosomal positions in early and mid-anaphases of all three cell types. Virtually all anaphases were measured, allowing a complete sampling of the mitotic segment. The x-axis distances measured between the early and mid-anaphase chromosomes were compared with different theoretical models of chromosomal separation on the mitotic ring (see Appendix ). The pooled x-axis distances measured between all of the homologous chromosomes in the lymphocyte, MRC-5, and CCD-34Lu early and mid-anaphases strongly fit the theoretical model for a random, but no other, distribution . The x-axis distances between the individual early and mid-anaphase chromosomes of these cell types predominately fit the random model, although some heterogeneity among these data sets was observed (see Appendix , Table IV ). The chromosomal separations measured in widely separated, late-anaphase rings between the homologues of the CCD-34Lu chromosomes X and 7 and MRC-5 chromosome 7 were random (see Appendix , Table I ), similar to the prometaphase rosettes. The nearest angles between the homologues of chromosome 7 and the X chromosome measured in MRC-5 (male) late-anaphase rings were highly variable, a finding inconsistent with fixed positions of these chromosomes on the late-anaphase rings (see Appendix , Table II ). Also, symmetrical positions were found for the same chromosomes in each daughter of the widely separated chromosomal masses, regardless of chromosomal mass morphology . This symmetry is quantified in Fig. 4 , which shows the correlation between angles measured in each daughter of 142 unselected, and consecutively measured, pairs of widely separated CCD-34Lu ( n = 72) and MRC-5 ( n = 70) chromosomal masses. In the figure, the x-axis coordinate of every point is the angle between two homologues measured in one chromosomal mass. The y-axis coordinate is the same angle measured in either the other daughter chromosomal mass of the pair or in a randomly selected chromosomal mass of the same cell type . The 214 pairs of angular measurements made in the daughter-paired chromosomal masses were highly correlated with each other (correlation coefficient = 0.788), whereas the randomly paired angles were not correlated (correlation coefficient = −0.087). The daughter-paired angular separations remained highly correlated after the morphologic separation into late-anaphase (54 pairs of measurements), telophase (58 pairs), and mixed/indeterminate (102 pairs) subgroups, with correlation coefficients of 0.856, 0.791, and 0.749, respectively. The randomly paired angles in these morphologic subgroups remained uncorrelated, with correlation coefficients of 0.010, −0.189, and −0.078, respectively. We have found several lines of evidence for a largely random assortment of chromosomal positions on the mitotic rings of three human cell types. The first question addressed was whether or not the rigorous >90° separation for all human homologous chromosomes reported by Nagele et al. for several cell lines could be confirmed and extended to other nontransformed human cells. We were unable to confirm this finding of >90° separation of homologous chromosomes in rosettes of the CCD-34Lu line, a cell type in which this phenomenon had been reported previously to occur , or in lymphocyte or MRC-5 rosettes. For all three cell types in our study, an equal number of rosette homologues were separated by <90° as by >90° . Also, the pooled x-axis distances between homologous early and mid-anaphase chromosomes of the lymphocytes, MRC-5, and CCD-34Lu cells strongly fit the random separation model and only weakly fit, or rejected, all other theoretical models of chromosomal separation . Finally, the individual angular separations measured in different pairs of late-anaphase rings between the homologues of CCD-34Lu chromosomes X and 7 and MRC-5 chromosome 7 were highly variable and thus incompatible with fixed chromosomal positions on the mitotic ring (see Appendix , Tables I and II ). These differing results between our study and Nagele's study are not due to variations in fixation, as the CCD-34Lu chromosomes 7 and X have random positions in both Carnoy- and paraformaldehyde-fixed rosettes (see Appendix , Table I ). The previously reported finding of widely separated homologous chromosomes led to the speculation that all human chromosomes were in the same fixed order on the mitotic ring and in interphase . In addition to our direct experimental evidence against widely separated and fixed chromosomal positions on the mitotic ring , there are strong theoretical arguments against Nagele's model of rigorously connected chromosomal positions being carried through interphase into subsequent mitotic and meiotic divisions . This model requires permanent interconnections between chromosomes, or some other mechanism, to maintain chromosomal spatial order. Although interphase chromosomes are connected to each other, if not by nucleotide strands , then by DNA–protein complexes , there is no evidence that such connections are permanent. The interphase positions of mammalian chromosomes are not static: Barr and Bertram showed that the position of the X chromosome shifted with electrical stimulation in postmitotic neurons. Shifts in interphase chromosomal positions have also been found in neurons from human epileptic cortex , in lymphocytes during different phases of the cell cycle , and in other cells with differentiation . Finally, although Dipteran homologues are paired in adult flies , histone gene repeats on the Dipteran chromosome 2 are randomly positioned in the nucleus during the first 13 embryonic cell cycles, and only subsequently pair in late embryos . It is difficult to imagine how such freedom of interphase chromosome movement, observed for a wide variety of cell types, can be reconciled with fixed and permanent connections between the chromosomes during interphase and on the mitotic ring. Also, if the fixed order of the relative positions of chromosomes on the mitotic ring was maintained from the initial fusing of parental haploid genomes into the next meiotic division, the random, Mendelian segregation of chromosomes could not occur. A simple mechanism can reconcile many of the conflicting results reported for relative chromosomal positions on the mitotic ring: some have shown loosely organized, or even random, chromosomal positions ; and others have shown nonrandom positions on the ring . Different chromosomes have discrete domains within the interphase nucleus . In 1885, Rabl suggested that the radial chromosomal positions on the mitotic ring during mitosis were a reflection of the relative chromosomal positions in the preceding interphase . The prophase movements of chromosomes support this view, as there are no wide shifts in the positions of the prophase chromosomes relative to each other as they move to the metaphase plate . The relative positions of the chromosomes to each other may vary in different interphase cells due to heterogeneity of nucleolus formation from cell to cell , specific transcription patterns induced in response to local differentiation signals , random drift, and possibly other types of chromatin–nuclear envelope interactions . The shifts in chromosomal positions due to differentiation or in response to external signals may be related to the coupling of actively induced genes to the mRNA processing machinery. Pre-mRNA transcription sites are preferentially associated with discrete pre-mRNA splicing domains . It is not clear whether the splicing domains are induced where transcription occurs, and/or whether actively transcribed genes move to these splicing domains . If the latter were true, differentiated or induced gene activity would determine gene, and possibly chromosome, location. In support of this occurring, γ-amino butyric acid, a powerful inducer of specific gene expression in pheochromocytoma cells, induces chromatin movement and kinetochore rearrangements in cultured mouse neurons . Also, estrogen induction of the vitellogenin gene family in male Xenopus laevis hepatocytes is associated with kinetochore rearrangements . In addition to our finding that seemingly all possible chromosomal arrangements may occur on the mitotic ring , several lines of evidence in our study also suggested that the relative positions of the chromosomes to each other on a given metaphase plate are transmitted into telophase with remarkable fidelity. First, the homologous centromeres clearly had symmetrical positions in the separating early and mid-anaphase chromosomal masses , ruling out chaotic shifts of chromosomal positions during early karyokinesis. Second, rings similar to those of the prometaphase rosettes and metaphases are present in many late-anaphases ; suggesting that the ring structure remains intact throughout karyokinesis. Finally, the centromeric positions measured in unselected, individual pairs of late-anaphase and telophase chromosomal masses are highly correlated , confirming earlier claims of symmetrical chromosomal positions in nonmammalian late-anaphases . All of these findings are consistent with the chromosomal positions on the mitotic plate being carried through anaphase into telophase. This finding of a permissive mitotic ring which transmits its relative chromosomal order into both daughter telophases suggests a mechanism by which the chromosomal organization of a given interphase nucleus is reestablished in its progeny. Specifically, the nonrandom chromosomal positions of a given interphase cell, induced by nucleolus formation, gene activation, differentiation, or other factors, may lead to similar, nonrandom chromosomal positions on the mitotic ring. This is strongly supported by the results of UV radiation experiments which showed that irradiation of small parts of G o /G 1 nuclei caused damage to only a few, usually nonhomologous, chromosomes that are later adjacent to each other on the mitotic ring , a finding consistent with adjacent interphase chromosomes injured by the irradiation ending up in close proximity to each other during mitosis. The symmetrical homologous chromosomes found in the daughter late-anaphase and telophase pairs in our and earlier studies can be simply explained by the carrying over of the relative chromosomal positions on the mitotic ring through anaphase into telophase. Taken together, these results suggest that the spatial chromosomal organization of the interphase nucleus is maintained from one generation to the next. In summary, there is a relatively random organization of chromosomal positions on the mitotic rings of human MRC-5 cells, CCD-34Lu cells, and lymphocytes, in contrast to a previous report of an invariable >90° separation of homologous human chromosomes on the mitotic ring . We also speculate that nonrandom chromosomal associations on the mitotic ring reported for other cell types may be due to the carrying over of nonrandom interphase chromosomal positions to the mitotic ring, and not to the mitotic ring apparatus selecting out a preferred radial chromosomal order before karyokinesis. Thus, our results show that a fixed order of chromosomal positions on the mitotic ring is not fundamental to, or necessary for, the mitotic segregation of human chromosomes, because human MRC-5 cells, CCD-34Lu cells, and lymphocytes go through mitosis quite smoothly. We also found that the relative positions of chromosomes on each metaphase ring seem to be carried through anaphase into telophase. | Study | biomedical | en | 0.999996 |
10189365 | Table I lists the genotypes of all yeast strains used in this study. Media for yeast growth and sporulation were as described , except as otherwise indicated. For experiments monitoring the loss of a nonessential chromosome fragment, adenine was added to 6 μg/ml minimal media to enhance the development of the red pigment in ade2-101 strains. For galactose inductions, strains were grown on solid medium containing 2% raffinose as the sole carbon source, and then transferred to solid medium containing 2% galactose, either with or without additional 2% raffinose. For experiments using liquid cultures, strains were grown in liquid medium containing 2% raffinose overnight, and galactose was added to a final concentration of 2% for the induction times indicated. To inhibit MT function in liquid cultures, nocodazole (NZ; Sigma Chemical Co. ) was added to 20 μg/ml and cultures incubated at 25°C for 2 h, or to 15 μg/ml at 30°C for 90 min. To inhibit MT function on solid medium, benomyl ( DuPont ) was added at 5, 10, and 20 μg/ ml as indicated. DMSO alone was added to the media as a control. All yeast transformations were done by the method of Ito et al. . Strains containing mutations in the gene of interest were mated to a ctf19 null strain to create the heterozygous diploids. These strains were sporulated, dissected, and cultured at 25°C. At least two independent diploid clones were analyzed for each mating. To test for conditional interactions, all double mutant spores recovered at 25°C were assayed for growth at higher temperatures, initially at 30°C and 37°C. Other temperatures were tested if there was an indication of a conditional effect at a permissive temperature to determine the lowest temperature at which lethality was observed. All synthetic lethal interactions indicated by the tetrad data were confirmed by plasmid shuffle. CBF3 mutant strains which demonstrated potential SL with a ctf19 null were mated to a ctf19 Δ 1::TRP1 or ctf19 Δ 1::HIS3 strain containing a wild-type copy of CTF19 on a URA3 -CEN plasmid (pKH7), sporulated and dissected. The bub (budding uninhibited by benzimidazole) and mad (mitotic arrest deficient) deletion strains were transformed with the appropriate wild-type gene ( BUB1 , BUB2 , BUB3 , or MAD2 ) on a URA3 -CEN plasmid, mated back to the ctf19 null strain, sporulated, and dissected. Double mutant spores recovered containing a URA3 -marked plasmid were streaked on plates containing 5-FOA, which does not allow growth of cells expressing URA3 . Therefore, only viable double mutants can grow on 5-FOA, and true synthetic lethals can not. Methods are as described previously . In brief, mutant strains were transformed under noninducing conditions, with overexpressing constructs containing either CTF13 (pKF88) or CTF19 (pKH21) reference genes under transcriptional control of a GAL1 promoter, and the corresponding vector, p415GEU2. Expression of the reference gene was induced on solid medium containing 2% galactose. Growth was compared directly for each mutant containing either an overexpression plasmid or the respective vector alone. To test for conditional effects, transformants viable at 25°C were also assayed at 30°C and 37°C. The chromosome missegregation phenotype of ctf19-58 was confirmed to be due to a single genetic mutation through genetic analysis after backcrossing this mutant strain to its wild-type parent (YPH277). The CTF19 gene was cloned by complementation of the ctf sectoring phenotype from a library containing 10–12-kb fragments of yeast genomic DNA inserted into a LEU2 -CEN vector (Spencer, F., and P. Hieter, unpublished results). Appropriate restriction fragments were used for subcloning the genomic DNA into pRS based vectors . A 2-kb MluI-NsiI fragment rescued the sectoring phenotype and was shown to contain the CTF19 gene by genetic linkage analysis. CTF19 was physically mapped to chromosome XVI by hybridization of a [ 32 P] labeled SalI-NsiI fragment to filters containing overlapping lambda and cosmid clones of the S . cerevisiae genome . This placed CTF19 on the right arm of chromosome XVI, 40–50 kb from CEN6 , distal to RAD1 . A complete deletion of the CTF19 open reading frame (ORF) was generated using PCR-mediated gene disruption . Oligonucleotides for PCR were synthesized as follows. OKH1 (5′-GTGTGATCTTGTT GATAC TAGGT CGCAAAGAACGCAAATAGATTG- TACTGAGAGTGCAC-3′) has a 40-bp homology to the sense strand upstream of the CTF19 ATG, followed by a 20-bp sequence from the plus strand of pRS vectors adjacent to the vector selectable marker. OKH2 (5′-GTTTAAGCAAGCCGTCCAGTTGGCAATGGCAAATGGAACACTGTGCGGTATTTCACACCG-3′) has a 40-bp homology to the antisense strand downstream of the stop codon followed by a 20-bp sequence from the minus strand of pRS vectors adjacent to the vector selectable marker. OKH1 and OKH2 can be used to incorporate any one of the markers from the pRS vectors ( URA3 , HIS3 , LEU2 , or TRP1 ) by PCR. These oligonucleotides were used to amplify either HIS3 from pRS303 or TRP1 from pRS304. The HIS3 PCR product was transformed into the haploid strain YPH877 and the diploid strain YJP57. The TRP1 PCR product was transformed in the haploid strain YPH1125 and the diploid strain YPH982 (Table I ). Gene replacement was confirmed in each case by Southern blot analysis. Plasmids containing CTF19 fused to the GAL1 promoter, either with or without an in frame E1 epitope tag, were constructed from the plasmid p415GEU1 and p415GEU2, respectively . A PCR strategy was used to place the second codon of CTF19 in frame with the ATG of p415GEU1 and p415GEU2, using a 5′ engineered Xho1 cloning site and a downstream engineered HindIII site . To avoid possible PCR errors, a wild-type PstI (bp 566 in ORF) HindIII (in multiple cloning site) genomic fragment was used to replace the 3′ end of the PCR-generated sequence in pKH20. When transformed into a ctf19 deletion strain containing pKF88 , both pKH20 and pKH21 were able to rescue the SDL phenotype. pKH20 and pKH21 were also able to stabilize a chromosome fragment in the ctf19 deletion strain when compared with the vector alone control on galactose containing media. To make an HA epitope-tagged construct, a PCR based site-directed mutagenesis method which utilizes Pfu polymerase was used to engineer a StuI site directly 3′ to the ATG of CTF19 in pKH6 (protocol from QuickChange™ site-directed mutagenesis kit; Stratagene), creating pKH27. Three tandem copies of the 9–amino acid HA epitope tag were amplified by PCR from the vector pSM492 (a gift from Susan Michaelis) with StuI sites engineered on the 5′ and 3′ ends. The triple HA tag was ligated into the StuI site engineered at the NH 2 terminus of CTF19 , and this epitope-tagged fusion construct was subcloned into pRS313 and pRS315, generating pKH31 and pKH32, respectively . These constructs were transformed into ctf19 Δ 1::TRP1 and shown to rescue the chromosome missegregation phenotype. The CTF19-3HA fusion was integrated into the genome by gamma integration . A 1.4-kb XhoI-SmaI fragment containing CTF19-3HA from pKH32 was subcloned into a HIS3 marked gamma integration vector, p679 , which is designed to direct an integration event at the leu2 Δ 1 locus on chromosome III. The resulting integrating construct, pKH35, was linearized at the NotI site, between the targeting sequences to the left (5′) and right (3′) of the leu2 Δ 1 locus, and transformed into a ctf19 Δ 1::TRP1 deletion strain , creating leu2 Δ 1::CTF19-HA3 . Integration at the leu2 Δ 1 locus was confirmed by colony PCR analysis, using primers OMB- leu2 Δ (5′-GTGTAGAATTGCAGATTCCC-3′, provided by M. Basrai) and T7. An integration event targeted correctly to the genomic leu2 Δ 1 locus results in a 550-bp product. Both the plasmid based and genome integrated versions of the CTF19-3HA epitope-tagged fusion produce the same 48-kD band by Western blotting and probing with anti-HA antibody, which is not seen in control strains containing a wild-type untagged copy of CTF19 . Colony color half sector analysis was performed as previously described . In brief, homozygous diploid strains containing a single SUP11- marked chromosome fragment were plated to single colonies on solid media containing a limiting amount of adenine (6 μg/ml), and grown at 30°C. The red pigment was allowed to develop at 4°C before scoring the sectoring phenotypes. Colonies scored as half-sectored were ≥50% red. A 1:0 missegregation event (chromosome loss) in the first division results in pink/red half-sectored colonies, whereas a 2:0 missegregation event (nondisjunction; chromosome gain) results in white/ red half-sectored colonies. Cells were processed for flow cytometry as previously described , with a few modifications. In brief, logarithmically growing cells were pelleted, resuspended in 0.2 M Tris, pH 7.5, containing 70% ethanol, and fixed overnight at 4°C. Cells were treated with 1 mg/ml RNase A at 37°C for 1 h. Proteinase K was added, and the cell suspension incubated at 55°C for 1 h. Cells were stained overnight at 4°C in 0.2 M Tris containing 3 μg/ml propidium iodide ( Sigma Chemical Co. ). Before being subjected to flow cytometry, cells were sonicated, using a Branson sonifier 450 on a setting of two for 2–5 s. Aliquots of cells fixed with 3.7% formaldehyde, as described for immunofluorescence, were stained with 300 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) to analyze nuclear morphology, and anti–α-tubulin decorated with fluorescein isocyanate, to analyze spindle morphology. For experiments comparing ctf13-30 and ctf19 Δ 1 single mutants to ctf13-30 ctf19 Δ 1 double mutants, cultures of wild-type (YPH501), ctf13-30/ctf13-30 , ctf19 Δ 1::HIS3 / ctf19 Δ 1::HIS3 , and ctf19 Δ 1::HIS3 / ctf19 Δ 1::HIS3 ctf13-30 / ctf13-30 were grown at 25°C to early log stage (∼10 6 cells/ml), then split at time = 0, keeping half of the culture at 25°C and shifting the other half to 37°C. Samples were taken at the start of the experiment (time = 0), and at 3 and 5 h after the temperature shift. For each time point, samples were assayed for viability, nuclear and spindle morphology, and cell DNA content by flow cytometry. Isolation of minichromosomes from yeast cells and assaying their ability to bind to MTs were performed as previously described . The methods used for chromatin immunoprecipitation and the PCR primers used to amplify both centromeric and noncentromeric test loci were as described in Meluh and Koshland . Purified HA mAb 12CA5 (Berkeley Antibody Co.) was added at either a 1:250 dilution (resulting in a 4 μg/ml final concentration), or 50 μl of HA antibody pre–cross-linked to CNBr–Sepharose beads was added to the chromatin solution. Crude anti-Mif2p rabbit antisera, provided by Pam Meluh (Carnegie Institute of Washington, Baltimore, MD), was used at a 1:250 dilution and served as a positive control for this assay. For NZ treatment experiments, NZ was added to logarithmically growing cells at 15 or 20 μg/ml final concentration, and incubated for 90 min at 30°C to completely depolymerize MTs before fixation. Ctf19p was localized by indirect immunofluorescence microscopy as described by Pringle et al. , with a few modifications. To visualize MTs, cells were fixed with 3.7% formaldehyde for 90 min at 30°C. To visualize Ctf19-HAp or Tub4p, cells were fixed for 60 min at 25°C. Primary antibodies were diluted in block solution (4% milk, 2% BSA, and 0.1% Tween in 1× PBS) as follows: 1:50 for anti–α-tubulin (YOL1/34, Serra Lab), 1:5,000 for anti-HA antibody (12CA5, Boehringer Mannheim Corp. ), and 1:500 for anti-Tub4p . Affinity-purified secondary antibodies (Cappel Research Products) were used at 1:1,000 dilution in block solution. YOL1/34 was detected with rhodamine conjugated goat anti–rat antibodies, anti-HA antibody was detected with fluorescein conjugated goat anti–mouse antibodies, and anti-Tub4p was detected with CY3 conjugated goat anti–rabbit antibodies (Jackson ImmunoResearch Laboratories, Inc.). When costaining for Ctf19-HAp and either α-tubulin or Tub4p, primary and secondary antibodies were applied in rounds, with anti-HA and goat anti–mouse applied first, to avoid background from secondary antibody cross-reactivity. Cells were examined using fluorescence microscopy with a Zeiss Axioskop fitted with UV, fluorescein isothiocyanate, and rhodamine/CY3 optics and a 100× objective. Digital images were captured using a cooled charged-coupled device (CCD) camera (Photometrix) and IPLabSpectrum software (Signal Analytics). An SDL screen was performed in which CTF13 was inducibly overexpressed in a subset of 12 potential kinetochore ctf mutants, which tested positive for the centromere transcription readthrough assay . Overexpression of CTF13 caused SDL in combination with four mutants within this set: ndc10-42 , ctf17-61 , ctf19-26 (YCTF26) , and ctf19-58 (YCTF58) . The chromosome missegregation ctf phenotype (visualized by the formation of red sectors in a white colony) of ctf19-58 was confirmed by genetic analysis to be due to a single nuclear mutation, and the corresponding gene was cloned by complementation of this phenotype . CTF19 was localized to the right arm of chromosome XVI using physical mapping methods. Nucleotide sequencing of the 2-kb CTF19 clone revealed a previously unidentified 1.2-kb ORF that encodes a protein of 369 amino acids with a predicted molecular mass of 40 kD. Database searching revealed no significant overall homology at the amino acid level. Protein motif searching (ProSite) revealed a putative leucine zipper beginning at amino acid 131 . The sequence and mapping data of CTF19 (YPL018W) was corroborated upon release of the complete sequence of the S . cerevisiae genome . ctf19-26 was confirmed to be an independent allele of CTF19 using four lines of evidence: first, the cloned CTF19 DNA complements the ctf19-26 sectoring phenotype; second, the ctf19-58 / ctf19-26 diploid exhibits a chromosome missegregation phenotype; third, when this diploid is sporulated, all spores which bear a chromosome fragment display the sectoring phenotype (Basrai, M., personal communication); and fourth, when ctf19-26 is crossed to a wild-type strain with the LEU2 -marked genomic CTF19 locus , the ctf phenotype always segregated away from the LEU2 marker. A complete deletion of the CTF19 ORF was generated using PCR-mediated gene disruption . The CTF19 ORF was replaced with two different marker genes, creating ctf19 Δ 1::HIS3 and ctf19 Δ 1::TRP1 . The deletion strains are viable, display no temperature conditional phenotypes, and exhibit a chromosome missegregation phenotype similar to that seen in strains containing either of the original mutant alleles, ctf19-58 or ctf19-26 . Upon overexpression of CTF13 under the control of a GAL1 promoter, SDL was observed in the deletion strain, comparable to that seen with ctf19-58 and ctf19-26 , albeit somewhat more pronounced. Strains carrying the ctf19 Δ 1 , ctf19-58 , or ctf19-26 mutations were tested for sensitivity to benzimidazole compounds. These agents cause depolymerization of MTs, and it has been observed that kinetochore mutants, as well as mitotic checkpoint mutants, are sensitive to compounds such as benomyl . All three strains are highly sensitive to 10 μg/ml of benomyl at 25°C, and slightly sensitive at the level of 5 μg/ml . Isogenic wild-type strains grow well with 10 μg/ml of benomyl at 25°C, and are sensitive to 20 μg/ml. As an interpretive note, the bub and mad mutants are also sensitive to 10 μg/ml of benomyl. Examination of possible cell cycle defects revealed that a ctf19 Δ 1/ctf19 Δ 1 mutant shows a G2/M accumulation with 2C DNA content in a logarithmically growing culture . Cytological analysis of these cells reveals an accumulation of large budded cells with the nucleus at or near the neck (29% in ctf19 Δ 1 versus 6% in wild-type). A similar G2 delay is seen in ctf13-30 , ndc10-42 , cep3-1/-2 , and skp1-4 kinetochore structural mutants at their nonpermissive temperatures. While there is an accumulation of large budded uninucleate cells, the number of telophase cells in the ctf19 null mutant remains similar to that seen for wild-type, 17% versus 16%, respectively. In contrast, the percentage of telophase cells in a ctf13-30 mutant at the nonpermissive temperature drops to 3%. This is consistent with the notion that ctf13-30 cells experience a strong arrest at G2/M at the nonpermissive temperature, whereas ctf19 null cells experience a shorter delay, most likely because the lesion is less severe. Chromosome missegregation in the ctf19 mutants was quantitated in homozygous diploid strains using colony color half sector analysis . Chromosome loss (1:0 segregation) results in a pink/red half-sectored colony, whereas nondisjunction (2:0 segregation) results in a white/red half-sectored colony. The rate of chromosome loss in a ctf19 Δ 1/ctf19 Δ 1 mutant is ∼100 times greater than that of wild-type, and the rate of nondisjunction is 60-fold higher than wild-type (Table II ). For ctf19 mutant alleles, the rates of chromosome loss and nondisjunction are 77- and 17-fold higher than wild-type for ctf19-58 / ctf19-58 , and 37- and 13-fold higher for ctf19-26/ctf19-26, respectively. Thus, both chromosome loss and gain are occurring in ctf19 mutant diploids, and the deletion strain is affected the most severely. Similar rates of chromosome loss and nondisjunction have been reported for essential kinetochore mutants ctf13-30 and skp1-4 . To explore a potential role of Ctf19p in kinetochore function, genetic analysis was used to look for synthetic phenotypes between ctf19 mutants and known kinetochore mutants. As our first genetic test, we used the SDL screen to assay the effect of overexpression of CTF19 in a wild-type background, and in strains containing mutations in each of the four subunits of the CBF3 kinetochore complex. CTF19 , the reference gene, was placed under a GAL1 promoter and this plasmid was transformed into a set of target mutants ( ndc10 , cep3 , ctf13 , and skp1 ). Growth was assessed upon galactose induction, as previously described . Results of this dosage study are summarized in Table III . SDL was seen when CTF19 was overexpressed in the background of two independent mutant alleles of NDC10 , ndc10-42 , and ndc10-1 , thus providing another genetic link between CTF19 and the kinetochore. Moreover, overexpression of NDC10 results in lethality in ctf19-26 and ctf19-58 mutants. The plasmid expressing CTF13 under a GAL1 promoter (pKF88) was also transformed into these mutant strains and viability assessed. Overexpression of CTF13 results in SDL in ndc10-42 (as previously described), and also in ndc10-1 at elevated temperature (30°C), as well as cep3-1 and cep3-2 (Table III ). To look for additional genetic interactions between CTF19 and CBF3 kinetochore mutations, double mutants were made between a ctf19 null mutant and the same CBF3 subunit mutations used in the dosage studies above. The heterozygous diploids were sporulated at 25°C and tetrads analyzed (Table IV ). To detect any conditional interactions, all double mutants recovered at 25°C were assayed for growth at successively higher semipermissive temperatures. The ctf19 Δ 1 ctf13-30 double mutant showed conditional SL at 30°C, which is lower than the nonpermissive temperature of ctf13-30 alone . ctf19 Δ 1 ndc10-42 double mutants could not be recovered at 25°C, although ctf19 Δ 1 ndc10-1 double mutants were viable at all permissive temperatures tested, demonstrating allele specific SL. ctf19 Δ 1 skp1-4 double mutants were conditionally synthetic lethal at 28°C, whereas ctf19 Δ 1 skp1-3 double mutants were viable at all permissive temperatures. This allele specificity is significant since the skp1-4 allele arrests in G2, and the skp1-3 allele arrests in G1 at the respective nonpermissive temperatures , suggesting that the interaction detected between CTF19 and SKP1 is within the G2/M phase of the cell cycle. Similarly, a conditional synthetic lethal interaction was detected between ctf19 Δ 1 and sgt1-3 , but not with sgt1-5 ( SGT1 is a suppressor of skp1-4 ; Kitagawa, K., personal communication). sgt1-3 arrests at G2/M, whereas sgt1-5 arrests at G1/S. Therefore, this allele specificity is analogous to the ctf19 Δ 1-skp1-4 interaction. Finally, both mutant alleles of CEP3 tested, cep3-1 and cep3-2 , were synthetically lethal with ctf19 Δ 1 at 25°C. Thus, CTF19 genetically interacts with all four components of the CBF3 CDEIII-binding complex. To address the specificity of these genetic interactions, a ctf19 Δ 1 strain was crossed to strains containing cbf1:: TRP1 , cse4-1 , or mif2-3 mutations, representing other proteins which function at the kinetochore, as well as tub1-1 or tub2 tubulin mutations. No synthetic lethal interactions were detected in any of the corresponding double mutants, except for ctf19 Δ 1 mif2-3 , which is inviable (Table IV ). Interestingly, Meluh and Koshland have shown that Mif2p interacts with CEN DNA primarily through CDEIII. Thus, the genetic interactions identified between CTF19 and the kinetochore appear specific for CBF3 and other CDEIII-associated components. The ctf19 Δ 1 ctf13-30 double mutant was a valuable reagent, because double mutants could be recovered at 25°C, but were conditionally synthetic lethal at 30°C. It has been shown that ctf13-30 alone causes initiation of the mitotic checkpoint at the nonpermissive temperature, resulting in delay at the G2/M point of the cell cycle . When ctf13-30 is combined with mad2 or bub1 mitotic checkpoint mutants, the cells do not arrest upon shift to the nonpermissive temperature, and rapid death ensues . In contrast, when ctf13-30 is combined with a ctf19 Δ 1 mutant, although a rapid loss in viability is observed upon shift to the nonpermissive temperature, the checkpoint appears to be intact judging by a G2/M delay visible in flow cytometry profiles at 25°C and 37°C (data not shown). This G2/M delay in the double mutant is accompanied by an accumulation of large budded uninucleate cells with short spindles. The inability of the ctf19 Δ 1 ctf13-30 double mutant to recover after exposure to the nonpermissive temperature suggests that there is a more severe structural aberration than in the ctf13-30 mutant alone. In light of the observations that the mitotic spindle checkpoint responds to impaired kinetochore function , we asked directly whether CTF19 genetically interacts with BUB1 , BUB2 , BUB3 , or MAD2 mitotic checkpoint genes. To do this, we again used the SDL assay. The CTF19 overexpression plasmid (pKH21) was transformed into mitotic checkpoint target mutants, bub1 Δ, bub2 Δ, bub3 Δ, and mad2 Δ. Upon induction of CTF19 on galactose-containing medium, growth was assessed as previously described. SDL was observed in the bub1 Δ and bub3 Δ strains, but no defect of growth was seen with mad2 Δ or bub2 Δ. To interpret these results, we also determined the effect of overexpressing CTF13 in the same target mutants, especially since the ctf13-30 mutation triggers the mitotic checkpoint resulting in a G2/M delay, as described above. When CTF13 was overexpressed in combination with bub2 Δ, bub3 Δ, and mad2 Δ, SDL was observed with bub3 Δ, but not with mad2 Δ or bub2 Δ. To test for synthetic lethal interactions, crosses were made between strains carrying a ctf19 null mutation and strains carrying either bub1 Δ, bub2 Δ, bub3 Δ, or mad2 Δ mutations (Table V ). A rad9 Δ mutation, previously characterized as defective in monitoring incomplete replication and DNA damage , was also tested. SL was seen when ctf19 Δ 1 was combined with bub1 Δ, bub3 Δ, or mad2 Δ, but not with bub2 Δ or rad9 Δ (Table V ). Bub1p, Bub3p, and Mad2p are all necessary for the initiation of the mitotic checkpoint pathway and are sufficient for shorter delays, as seen with low levels of NZ, whereas Bub2p is required for maintaining longer delays, as seen with high levels of NZ . These synthetic dosage lethal and synthetic lethal genetic interaction results are consistent with the notion that ctf19 mutations invoke a mild premitotic delay, which does not require Bub2p. Taken together, these genetic studies strongly suggest that Ctf19p plays a structural role at the kinetochore. Sucrose gradient sedimentation analysis revealed that Ctf19-HAp sediments with an S value of 20 (data not shown). This suggests that Ctf19p may be part of a large protein complex in the cell. There is no evidence that Ctf19p is part of the CBF3 CDEIII-binding complex, as Ctf19p is not required for the normal CEN DNA bandshift in vitro, and antibodies to an epitope-tagged Ctf19p do not result in a supershift of the CBF3- CEN DNA complex . In addition, coimmunoprecipitation experiments using crude yeast cell extracts were unable to detect a direct interaction between Ctf19p and CBF3 proteins. Knowing that Ctf19p is not a part of the CDEIII-binding complex detected by CEN DNA bandshift in vitro, we investigated other ways in which it could be involved in kinetochore function. One possibility is that Ctf19p plays a role in binding of MTs to kinetochores. To test this hypothesis, an in vitro assay was used which measures the ability of yeast centromeres to bind to MTs . In this assay, minichromosomes containing a centromere are introduced into wild-type or mutant strains, and purified chromatin is prepared. Taxol-stabilized bovine MTs are added to the lysate and sedimented. The supernatant and pellet are separated, DNA from each fraction is extracted, and the relative amount of the minichromosome in each fraction is determined. In wild-type cultures with wild-type centromeres on the minichromosomes, the minichromosomes sediment with the MTs. CEN DNA mutations in CDEII and CDEIII abolish the centromere's ability to bind MTs . Similarly, trans-acting mutations in CEN -binding proteins also inhibit the ability of the minichromosome to bind MTs, exemplified by the cep3-1 mutant . Two independent alleles of CTF19 were tested in this assay, ctf19-58 and ctf19-26 , and both were shown to exhibit a dramatic defect in centromere-dependent binding of minichromosomes to MTs . Therefore, knowing that Ctf19p interacts genetically with the CBF3 complex, is important for binding MTs to centromeres of minichromosomes, and sediments with an S value consistent with being part of a protein complex, we next investigated whether Ctf19p is involved in a higher order centromere complex. To examine whether Ctf19p physically interacts with a kinetochore macromolecular complex, we used an in vivo cross-linking and immunoprecipitation strategy . Formaldehyde cross-linked chromatin (2 h fixation) prepared from an epitope-tagged CTF19-3HA strain and a wild-type untagged control strain were sonicated to shear the DNA and immunoprecipitated with anti-HA antibody. After reversing the cross-links, the presence of specific DNA sequences in the immunoprecipitates was assessed by PCR analysis. Mif2p, which was previously shown to associate with CEN DNA in vivo , served as a positive control for this assay. Two CEN DNA sequences were tested, CEN3 and CEN16 , and both were found to be present in the Ctf19-HAp immunoprecipitate, but not in the untagged control strain or in the mock-treated control . The anti-Mif2p immunoprecipitate also contained these CEN DNA sequences. To test if the interaction detected is specific for CEN DNA, two noncentromeric loci, PGK1 and HMRa , which are AT-rich intergenic regions located on yeast chromosome III, were analyzed. Both of these sequences were not found in the Ctf19-HAp immunoprecipitate, as was the case with the Mif2p positive control . Thus, as predicted by the numerous genetic interactions with kinetochore protein components, Ctf19p specifically associates with the centromere in chromatin preparations. It is plausible that the association of Ctf19p with the centromere is transient and contact may be made with kinetochore proteins upon MT attachment. To test whether MTs are necessary for Ctf19p to associate with the centromere, the chromatin immunoprecipitation assay was performed with strains grown in the presence of NZ, a MT depolymerizing agent. Results from this experiment show that, as with Mif2p and Ndc10p (not shown), Ctf19p specifically immunoprecipitates CEN DNA, and not noncentromeric loci, even in the presence of NZ . These data further support the conclusion that Ctf19p is in fact part of a centromere–protein complex. The localization of Ctf19p in yeast cells was determined by indirect immunofluorescence using an HA epitope fusion construct (pKH32). To avoid potential copy number effects, the CTF19-3HA fusion was integrated into the genome at the leu2 Δ 1 locus . For immunofluorescence studies, an asynchronous population of cells from strains containing CTF19-3HA or an untagged control were formaldehyde-fixed, and stained with anti-HA antibody and anti–α-tubulin, which stains spindle MTs. The HA antibody detected a bright dot at the vertex of the MTs in interphase cells, and at the ends of the spindle in mitotic cells . This staining pattern, reminiscent of SPB staining, is similar to that seen with other centromere proteins, including Ndc10p , Mif2p , and Cse4p , and is consistent with the notion that centromeres display a nonrandom localization throughout the cell cycle, clustering near the SPB during interphase (G1) and late mitosis . A key difference between the staining pattern observed for Ctf19-HAp and those reported for other centromere proteins occurs during early mitosis. Both Ndc10p and Cse4p are reported to stain the spindle MTs, evident as a short bar, in cells with short spindles. Ctf19-HAp staining, however, resolves into two distinct foci even in cells with short spindles. To examine the specificity of this localization, we obtained antibodies against Tub4p (provided by L. Marschall, Stanford, CA), which served as an SPB marker . Tub4p, the γ-tubulin homologue in S . cerevisiae , is part of a complex that localizes to the inner and outer plaques of the SPB , where nuclear and cytoplasmic MTs are organized, respectively. Costaining experiments revealed that Ctf19-HAp localizes to the same region as Tub4p . Further analysis of cells stained for both Ctf19-HAp and Tub4p revealed that the Ctf19-HAp signal appears to be just interior to (on the nucleoplasmic side of) the Tub4p signal in mid-mitotic cells with short spindles . This observation was examined in more detail by measuring the distance between the Ctf19-HAp signals and Tub4p signals in individual cells. The average distance between Ctf19-HAp signals for 50 mitotic cells was 1.5 μm ± 1.1, whereas the comparable distance between Tub4p signals was 1.8 μm ± 1.1. These measurements correspond to an average distance of 0.2 μm between Ctf19p and Tub4p in mitotic cells. To interpret the significance of this difference, it should be considered that Tub4p resides on both nuclear and cytoplasmic surfaces of the SPB and that the fluorescent signal observed for Tub4p is an average of both signals. Therefore, the distance from the Ctf19-HAp signal to the nuclear side of the SPB may be less than indicated by these measurements. These data suggest that Ctf19p localizes near the SPB, but, as expected, is not likely to be an integral component of the SPB. Consistent with this hypothesis, we were unable to demonstrate a direct interaction between Ctf19-HAp and Tub4p by coimmunoprecipitation (data not shown). It is possible that Ctf19p is part of a complex which resides at the nucleoplasmic surface of the SPB and transiently interacts with kinetochores. However, it is more likely that the staining pattern observed for Ctf19-HAp corresponds to a centromere localization, as noted above for other kinetochore proteins, and is thus compatible with data implicating a kinetochore function for Ctf19p. To test if we could differentiate between these two hypotheses, we asked whether Ctf19p requires MTs for its localization. Assuming that kinetochores are tethered to the SPB in a MT-dependent manner , cells were treated with 15 μg/ml NZ (a concentration that depolymerizes all MTs) and examined by immunofluorescence. For proteins that are known to be integral components of the SPB, localization is unchanged by the presence of NZ . Fluorescent in situ hybridization analysis has revealed that centromere localization is, however, changed in the presence of NZ as they are no longer clustered . It should be noted that, although NZ and other benzimidazole drugs depolymerize most of the MTs, it is possible that a small amount of the polymer which is undetectable by immunofluorescence remains . Interestingly, Ctf19p localization in most cells remained near the SPB, visualized as one (∼66% of cells) or two (∼27% of cells) brightly staining dots . This compares to 69% and 31%, respectively, in cells not treated with NZ. However, ∼7% of the cells (out of 500 cells analyzed) contained three or four discrete signals for Ctf19-HAp. There were also cells observed in which two signals were seen for Ctf19-HAp, but only one signal was seen for Tub4p. These observations indicate that the majority of Ctf19p remains near the SPB in the absence of MTs, however, a portion of the protein in some cells has an altered localization in the presence of NZ. These results may indicate that Ctf19p is present both at the centromere and near the nucleoplasmic surface of the SPB. Whether or not these localization data are suggestive of a function for Ctf19p at the SPB, or merely a consequence of the limitations of the assay, has yet to be determined. As a preliminary investigation into a potential role for Ctf19p at the SPB, we tested for genetic interactions between CTF19 and several genes encoding either integral SPB proteins or proteins involved in SPB duplication. No synthetic lethal interactions were detected in any double mutants created between ctf19 Δ 1 and mps2-1 , ndc1-1 , cdc31-1 , spc42-10 , spc110-1 , tub4-32 , or tub4-34 (data not shown). In comparison to the unequivocal genetic interactions detected between CTF19 and the genes encoding the CBF3 kinetochore components, we conclude that the function of Ctf19p lies at the kinetochore. We also tested for a genetic interaction between CTF19 and NDC80 , which encodes a spindle- and pole-associated protein. Interestingly, a conditional synthetic lethal effect was detected in ndc80-1 ctf19 Δ 1 double mutants at 28°C. This genetic interaction is allele specific, as no conditional SL was detected in ndc80-2 ctf19 Δ 1 double mutants. NDC80 mutants display phenotypes very similar to a temperature-sensitive mutant of NDC10 , which encodes a component of the CBF3 kinetochore complex . Although the phenotypes and immunofluorescent staining pattern of Ndc80p are consistent with localization to the kinetochore, no genetic interactions have been detected between NDC80 and CBF3 kinetochore components . These authors have concluded that an indirect interaction may exist between Ndc80p and the kinetochore. It is quite possible that such an indirect interaction may occur through Ctf19p. We have identified and characterized the CTF19 gene of S. cerevisiae , and show that it encodes a novel protein which is required for faithful chromosome transmission and efficient binding of centromeres to MTs, associates with a centromere DNA complex in vivo, and localizes to the SPB region. Previously, two uncharacterized mutations, ctf19-58 and ctf19-26 , were classified as putative kinetochore mutants by an in vivo centromere transcription readthrough assay . ctf19 mutants were further implicated in kinetochore function by our SDL screen , in which overexpression of the CTF13 reference gene, encoding an essential CBF3 kinetochore protein, resulted in lethality in the context of the ctf19-58 and ctf19-26 mutations. The phenotypes described here for ctf19 mutants, including chromosome missegregation, increased sensitivity to benomyl, and a G2/M accumulation of logarithmically growing cells, are all consistent with a defect in kinetochore function. Extensive genetic analysis revealed that CTF19 exhibits strong genetic interactions with both kinetochore structural mutants and mitotic checkpoint mutants. Genetic interactions (SDL, SL, or both) were detected between the ctf19 null mutation and mutant alleles of all four subunits of the CBF3 kinetochore complex, as well as with Mif2p, which has been shown to interact with CEN DNA in a CDEIII-specific manner . We also demonstrate that cells defective in, or overexpressing Ctf19p require a functional mitotic checkpoint pathway, as do cells defective in, or overexpressing Ctf13p. The extent and specificity of these interactions strongly implicates Ctf19p as playing a role in kinetochore structure or function. Consistent with the numerous genetic interactions linking Ctf19p to the kinetochore, as well as the sedimentation data, we demonstrate that Ctf19p associates specifically with centromeric DNA in vivo through formaldehyde cross-linking followed by chromatin immunoprecipitation. Two known kinetochore proteins, Ndc10p and Cbf1p, as well as two implicated kinetochore proteins, Mif2p and Cse4p, have been shown to cross-link wild-type CEN DNA in vivo . Ndc10p and Mif2p both demonstrate a specific dependence on the integrity of CDEIII . A similar specificity of interaction between Ctf19p and CDEIII is described by Lechner and colleagues, who have independently identified Ctf19p as part of a complex of proteins that interacts with CBF3 . These data are consistent with genetic interactions reported here, which are also specific for CBF3 components. The fact that Ctf19p is able to specifically cross-link CEN DNA does not differentiate between a direct and indirect association. Ctf19p is not a subunit of the CBF3 complex , and is not necessary for the gel mobility shift seen with the assembled kinetochore complex on a CEN DNA fragment (data not shown; Lechner, J., personal communication). Therefore we propose that Ctf19p interacts indirectly with CEN DNA, likely through interactions with the CBF3 complex, or perhaps through a larger macromolecular complex whose assembly is initiated by recruitment of CBF3 to CDEIII . Given the ability of Ctf19p to cross-link to CEN DNA, and the defect seen in the ability of ctf19 mutants to bind centromeres of minichromosomes to MTs, Ctf19p would be a good candidate for a factor which interacts with CBF3 and is necessary to form active spindle MT-binding complexes. The fact that Ctf19p is able to associate with centromeric DNA in the presence of NZ suggests that MTs are not required for this association, and places Ctf19p at the kinetochore instead of on the spindle. Similar results were observed for Mif2p and Ndc10p. We are currently testing whether Ctf19p is required for attachment of reassembled kinetochore complexes in vitro to polymerized MTs. The immunolocalization pattern of Ctf19p is consistent with kinetochore proteins, which is strongly supported by the genetic and biochemical data presented here. Other proteins which have been biochemically placed at the centromere, including Ndc10p, Mif2p, and Cse4p, show immunofluorescent staining patterns similar to Ctf19p. The SPB staining pattern observed in G1 cells and in late mitotic cells could be due to the effect of centromere clustering, a phenomenon seen in higher eukaryotes and demonstrated in yeast by fluorescent in situ hybridization . Studies on cell cycle dependent centromere positioning in S . cerevisiae reveal that in G1 cells, centromeres are loosely clustered around the SPB, similar to that reported for fission yeast and mammalian cells . In mitosis before anaphase (mid M), centromeres are centrally localized within the nucleus, away from the SPB, reminiscent of metaphase in larger eukaryotic cells. In anaphase and telophase cells, centromeres are clustered tightly and proximal to the SPBs. One caveat to interpreting the Ctf19p localization as consistent with it being a CEN binding protein is that, unlike Ndc10p and Cse4p , Ctf19p does not display spindle staining, as may be expected for centromere associated proteins when kinetochores are forming bipolar attachments to the spindle MTs. The absence of spindle staining for Ctf19p may reflect limitations of the immunofluorescence assay, as other kinetochore proteins, including Ctf13p and Cep3p, have not been visualized in the cell by traditional immunofluorescent techniques, or it may reflect a unique role for Ctf19p in mitosis, perhaps functioning to tether kinetochores to the SPB. Given the prominent role of anaphase B in sister chromatid separation in budding yeast , Ctf19p may be important in stabilizing the link between kinetochores and each SPB during spindle pole separation. The results of immunofluorescence with Ctf19-HAp in the presence of NZ suggest that at least some amount of Ctf19p localizes adjacent to the SPB, as opposed to Ctf19p residing solely at the kinetochores, because the positioning of kinetochores near the SPB is believed to be dependent on MTs. In the presence of NZ, it has been observed that centromeres are no longer clustered, but rather disperse throughout the nucleus , presumably because MTs are no longer present for the kinetochores to remain attached to the SPB. However, the procedures used to fix and process nuclei for in situ hybridization in yeast may be more disruptive than that used for immunofluorescence, and theoretically could contribute to an aberrant delocalization of centromeres after treatment with NZ. Although Ctf19-HAp does maintain a discrete localization in the presence of NZ, we did observe a few examples (∼7% of cells analyzed) in which more than two fluorescent signals were seen for Ctf19-HAp which did not correlate with a Tub4p signal. We reason that, because a small polymer of MTs may still be present at the face of the SPB even after NZ treatment , Ctf19p may be part of a complex that is peripheral to the SPB. The localization of this complex may be less stable in the presence of MT depolymerizing agents, accounting for the small population of cells that exhibit more than two Ctf19-HAp signals. Wigge et al. have identified several new spindle pole and spindle-associated proteins through analysis of purified spindle preparations with MALDI mass spectrometry. Ndc80p is of particular interest because it stains spindles as well as poles, and ndc80-1 mutants display phenotypes similar to yeast kinetochore mutants, including chromosome missegregation and anaphase defects. In addition, Ndc80p is a potential homologue of human HEC protein, which localizes to the centromere. However, no genetic interactions were detected between NDC80 and three of the CBF3 components tested. Ndc80p partially copurifies with the factors which bind kinetochores to MTs , but is absent from the final fraction . Since Ctf19p interacts with the centromere, both genetically and biochemically, and it genetically interacts with Ndc80p, we propose that Ctf19p provides a link between the mitotic spindle and the kinetochore in budding yeast. | Study | biomedical | en | 0.999997 |
10189366 | Antiserum to synthetic peptides of segments of 10 kD (MESVPEPRPSEWDK), 22–24 kD (GVLLTAQTITSETPSSTTTTKITKC, exon 19) domain, or to the recombinant HP of 4.1R (anti-HP 4.1) was produced. Antisynthetic peptide antibodies were prepared and purified as described earlier . The DNA fragment containing the coding sequence for the 209 amino acids of HP of 4.1R was fused in frame to GST by using pGEX-2T vector, expressed in Escherichia coli , and purified on glutathione-Sepharose 4B beads according to the manufacturer's recommendations ( Pharmacia Biotech Inc. ). Aliquots of GST-HP 4.1 fusion protein were mixed with complete Freund's adjuvant and injected subcutaneously into New Zealand White male rabbits. 4 wk later the rabbits were given a booster dose of incomplete adjuvant. Sera were collected by bleeding through ear vein and were cleared by centrifugation. The sera were purified on immunoaffinity columns using resins covalently cross-linked to the fusion peptide used for immunization. The purified antibodies were eluted from the columns with 0.1 N acetic acid, neutralized, and dialyzed against 0.1 M borate buffer, pH 8.0. Anti-NuMA antibodies (raised in rabbit) used in this study were previously characterized . In some experiments, an anti-NuMA mAb (Oncogene) was also used. The anti–Na + -K + -ATPase α1 polyclonal antibody (Ab) used was described previously . Antidynein mAb (intermediate chain, clone 70.1), anti-actin mAb, antispectrin (α and β) mAb, and anti–γ-tubulin Ab were purchased from Sigma Chemical Co. Anti-p150 glued mAb (the largest subunit of dynactin) was purchased from Transduction Laboratories. Antipericentrin Ab and anticalmodulin mAb were purchased from BAbCO and Zymed Laboratories, Inc. , respectively. cDNAs were generated by restriction digestion of 4.1R cDNAs or by PCR using an amplification kit ( Promega Corp. ) and a thermocycler (PCR System 480; Perkin-Elmer Corp. ). For the full-length (135-kD) 4.1R construct, the SmaI and BglII fragment from pTM-Full was subcloned into the SmaI and BamHI site of pAS2-1. For subcloning the 80-kD 4.1R or its different domains, DNA sequences were PCR amplified from 135 kD/PTM-Full or 80 kD/pEry-1,2,3 using custom oligonucleotide primers (Genosys Biotechnologies) designed with SalI and PstI sites at their 5′ or 3′ ends, respectively. The primers designed for generation of 4.1R domains were as follows: Hp/pAS2-1 (47–69/651– 673), 30 kD/pAS2-1 , 16 kD/pAS2-1 , 10 kD/pAS2-1 , and 22–24 kD/pAS2-1 . The amplified cDNAs were digested with SalI and PstI, purified, and cloned into SalI and PstI sites of Gal4 DNA-binding domain vectors, pGBT9 or pAS2-1, by standard methods. Different NuMA constructs containing the NuMA cDNA corresponding to NH 2 -terminal domain (308–878), coiled-coil domain , or COOH-terminal domain were cloned in frame with Gal4 transactivation domain in vector pACTII. The 135-kD/GST fusion construct was generated by subcloning the EcoRI and SalI insert of 135 kD/ pAS2-1 into EcoRI and SalI sites of pGEX-6P1 ( Pharmacia Biotech Inc. ). For 80 kD/GST and its different domains in GST vector pGEX-6P1, custom primer sets with the same sequences used for Gal4 DNA-binding domain (Gal4-BD) constructs, except with EcoRI or SalI at their 5′ or 3′ ends, were used to amplify the corresponding cDNA sequences, digested with EcoRI and SalI, and were subcloned in frame into pGEX-6P1. The 135 ++ kD/GFP construct was generated by cloning a human 4.1R cDNA containing all exons except exons 3, 14, 15, 17a/a′ and 17b in frame into KpnI and BamHI sites of pEGFP-C1 ( CLONTECH Laboratories). NuMA/TOPO constructs containing sequences corresponding to 5356–6561 (NuMA1/TOPO), 5356–5929 (NuMA2/TOPO), or 5752–6561 (NuMA3/TOPO) of NuMA were amplified with addition of the Kozak consensus sequence and the translation initiation site (GCCACCATG) incorporated into the 5′ end and a stop codon at the 3′ end. The amplified fragments were cloned into pCR ® -Blunt II-TOPO vector (Invitrogen Corp.). To ensure correctness of the reading frame, the 5′-junction of each construct was sequenced using a sequencing kit (sequenase v. 2.0; Amersham Corp. ). Deletion constructs representing different exons of the 22–24-kD domain of 4.1R were derived from 22–24 kD/pAS2-1 by using custom oligonucleotide primers as above, and were cloned into SalI and PstI sites of pGBT9. The primers used were as follows: exons 17–21/pGBT9 , exon 18–21/pGBT9 , exon 18–20/pGBT9 , exon 18–19/pGBT9 , exon 19/pGBT9 , exon 20/pGBT9 , exon 21 , and exon 20–21 . The construct exon 19–21/pGBT9 was made by a two-step PCR using the primer set and for the first round of amplification. The primary PCR products were annealed and the second round of PCR was done with the 5′and 3′ primers . The constructs exon 21 (amino acids 762–775)/pGBT9 and exon 21 (amino acids 745–761)/pAS2-1 were derived from 22–24 kD/pAS2-1 by using the SmaI and StyI sites, respectively. Different deletion constructs of NuMA were derived from NuMA 1476–2115/pACT2 or NuMA 1697–2115/ pACT2 . The plasmids were digested with BglII, the fragment of interest was purified, digested with the available restriction enzyme at the 3′ site of interest, and blunt-ended. Afterwards, the cDNA fragments were digested with EcoRI, purified, and cloned into pACT2 at the EcoRI and blunt-ended XhoI sites. The NuMA 1788–1832/pACT2 construct was derived from NuMA 1697–2115/pACT2 using custom oligonucleotide primers designed with EcoRI and XhoI site at their 5′ or 3′ ends, respectively, as above. The authenticity of all deletions was confirmed by dideoxy sequencing in both directions. The Gal4-based MATCHMAKER two-hybrid system II of CLONTECH Laboratories, Inc. was followed for the yeast two-hybrid assays. Plasmid vectors, pAS2-1 or pGBT9, and pACT2, encoding the Gal4-BD and Gal4-activating domain (Gal4-AD), respectively, were used to express hybrid proteins. To screen for proteins that interact with 4.1R in yeast two-hybrid system, a human brain cDNA library in Gal4-AD vector pACT2 was screened using either the full-length 4.1R (135-kD) or the 22–24 kD of 4.1R cloned into Gal4-BD vector (see plasmid construction section for details) as the bait. Positive clones were tested further for specificity by cotransformation into Y190 either with 4.1R 135 kD/pAS2-1, 22–24 kD/ pAS2-1, or with pAS2-1 alone. DNA from positive clones were isolated, the Gal4-AD plasmids were recovered in bacteria strain HB 101, and sequenced by dideoxy method as above. For domain mapping, plasmids carrying respective inserts fused to Gal4-BD or Gal4-AD were cotransformed into yeast, and were assayed for β-galactosidase activity on nitrocellulose filters as described in CLONTECH Laboratories' manual. The in vitro transcription/translation of NuMA/TOPO was performed using the TNT ® coupled (reticulocyte lysate system; Promega Corp. ) in the presence of [ 35 S]methionine to radiolabel newly synthesized proteins. Equal amounts of the labeled NuMA proteins were incubated with affinity-purified GST/4.1R fusion proteins coupled to glutathione-Sepharose beads for 1 h at 4°C in binding buffer (50 mM potassium phosphate, pH 7.3, 150 mM NaCl, 2.7 mM KCl, 4 μg/ml aprotinin, 4 μg/ml leupeptin, 4 μg/ml antipain, 12.5 μg/ml chymostatin, 12 μg/ml pepstatin, 130 μg/ml ε-amino caproic acid, 200 μg/ml p -NH 2 -benzamidine, and 1 mM PMSF). After incubation, the immobilized NuMA-4.1R complex was washed five times with binding buffer in the presence of 1% Triton X-100, once with the binding buffer plus 1% Triton X-100, 2 M urea and 100 mM glycine, and once with the binding buffer. The bound protein complex was subjected to analysis on a 15% or 18% SDS-PAGE, treated with Enlightning (NEN Life Science), and visualized by fluorography. HeLa (ATCC CCL 2, human cervix epitheloid carcinoma) and MDCK cells were grown in DME supplemented with 10% heat-inactivated FBS ( GIBCO BRL ). The cDNA encoding the full-length 4.1R in pEGFP-C1 vector (135 ++ kD/GFP) or the vector pEGFP-C1 was transiently transfected into HeLa cells using LipofectAmine reagent following the manufacturer's procedure ( GIBCO BRL ). The 135 ++ kD/GFP cDNA contained all of the known 4.1R exons except exon 3 and the tissue-specific exons 14, 15, 17a/a′, and 17b. Cells in a 100-mm 2 dish were transfected for 16 h, the GFP- or 135 ++ kD/GFP– transfected cells were sorted using a FACStar ® cell sorter to avoid the untransfected cell population overgrowth, and the transfected cells were collected. The sorted cells were plated on poly- d -lysine–coated coverslips and the samples were taken daily for 5 d, fixed, and processed for immunofluorescence staining. The transfected cells were immunofluorescence-stained using Texas red–conjugated anti–mouse IgG, anti-NuMA mAb, and TO-PRO-3 (for DNA staining), and were examined for the presence of 135 ++ kD/GFP fusion protein, Texas red, and Cy 5 fluorescence in individual cells. Highly enriched mitotic HeLa cells were prepared by synchronization of the cells at G1/S boundary of the cell cycle by double thymidine block . Cells were grown in complete medium for 4 h and the mitotic population was enriched by addition of 0.25 μg/ml of nocodazole for 6 h. Afterwards, cells were washed twice with complete medium, allowed to grow for 90 min in complete medium, and were collected by mitotic shake-off. Expression of 4.1R and NuMA at protein level in MDCK cells was documented by Western blotting. Cytoplasmic-, nuclear-, and nuclear matrix-protein fractions were prepared as described . The protein contents were determined using a protein determination kit ( Pierce Chemical Co. ). 20 μg of protein from each fraction was analyzed on 8–12% SDS-polyacrylamide gels (National Diagnostics Inc.). NuMA and 4.1R were detected by Western blotting using 1:500 dilution of anti-HP 4.1 or anti-NuMA antibodies as described earlier except that HRP conjugated goat anti–rabbit IgG and an ECL detection kit ( Amersham Corp. ) were used. Coimmunoprecipitation of 4.1R and NuMA was performed using MDCK nuclear extracts. Nuclei were purified as described , resuspended in coimmunoprecipitation (IP) buffer (25 mM Tris-HCl, 100 mM NaCl, 1 mg/ml BSA, 0.2 mM EDTA, 5 mM iodoacetamide, 0.05%[wt/vol] SDS, 4 μg/ml aprotinin, 4 μg/ml leupeptin, 4 μg/ml antipain, 12.5 μg/ml chymostatin, 12 μg/ml pepstatin, 130 μg/ml ε-amino caproic acid, 200 μg/ml p -amino-benzamidine, and 1 mM PMSF, pH 7.5), and given 20 strokes in a tight-fitting glass homogenizer. The nuclear homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was used for coimmunoprecipitation. Preimmune serum (rabbit) equivalent to 30 μg IgG was added to 1 mg of nuclear proteins and incubated for 2 h at 4°C, followed by the addition of 200 μl of a 50% suspension of protein A–Sepharose 6 MB ( Pharmacia ) in IP buffer for 2 h at 4°C. The supernatant was collected by centrifugation at 5,000 g for 5 min at 4°C and equally split into five tubes. Preimmune serum, affinity-purified anti-HP 4.1 Ab, anti-NuMA mAb, mouse IgG, or anti-p53 mAb containing 4 μg IgG were added to different tubes and incubated for 2 h at 4°C. Protein A–Sepharose 6 MB (100 μl of a 50% suspension) was added and incubated as above. For immunoprecipitation using mitotic HeLa extracts, cells were homogenized as above in IP buffer containing 170 mM NaCl, 2% Nonidet P-40, 0.1% (wt/vol) SDS, and 5 mM iodoacetamide. After centrifugation at 10,000 g for 10 min at 4°C, the supernatant was used for immunoprecipitation using 4 μg each of anti-NuMA mAb, anti-HP 4.1 Ab, antidynein mAb, anti-p150 glued mAb, anti-p53 mAb (an irrelevant antibody), mouse IgG or preimmune serum (rabbit), as above. After incubation with protein A–Sepharose, the samples were centrifuged for five min at 5,000 g at 4°C, and washed 5–10 times in 900 μl of IP buffer containing 1% Nonidet P-40 and 5 mM iodoacetamide. The samples were resuspended in 60 μl of SDS sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% [wt/vol] SDS, 5% 2-mercaptoethanol, and 10 μg/ml bromophenol blue, pH 6.8) and heated at 98°C for 5 min. The samples were centrifuged at 10,000 g for 10 min at 4°C and the supernatants were fractionated on 7–10% SDS-polyacrylamide gels. Transfer of proteins to nitrocellulose membrane and immunodetection of 4.1R or NuMA were carried out as described above. Some chemiluminograms were scanned using the Adobe Photoshop software (Adobe Systems, Inc.) and the protein bands of interest were quantitated by using the NIH Image software for the Apple Macintosh computer. In some cases, the gels were stained with the GelCode ® Blue Reagent or the GelCode ® SilverSNAP™ ( Pierce Chemical Co. ). MDCK or HeLa cells were grown on poly- d -lysine–coated glass coverslips. Coverslips were washed twice with PBS and immersed in a microtubule stabilizing buffer (4 M glycerol, 100 mM Pipes, pH 6.9, 1 mM EGTA, and 5 mM MgCl 2 ) for 2 min at room temperature and fixed in 2% paraformaldehyde, 0.4% glutaraldehyde, 90 mM Pipes, pH 6.8, 1 mM EGTA, and 5 mM MgCl 2 for 5 min at room temperature. Cells were washed three times with TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% albumin) and permeabilized with TBS containing 0.5% Triton X-100 for 5 min. Subsequently, the cells were washed three times with TBS containing 10 mM glycine. Cells were blocked with TBS containing 10% normal goat serum for 30 min at room temperature. For single staining, cells were either incubated with anti-HP 4.1 antibody (diluted 1:50) or anti-NuMA antibody (diluted 1:100) in TBS for 1 h at room temperature. Cells were washed in 10 mM glycine/TBS and incubated with goat anti–rabbit IgG conjugated to FITC (for anti-HP 4.1) or donkey anti–rabbit IgG conjugated to Texas red (for NuMA) for 1 h at room temperature followed by four washes in 10 mM glycine/TBS. When double staining was desired, after the first set of staining, cells were blocked with TBS containing 10 μg/ml goat anti–rabbit Fab fragment followed by four washes in 10 mM glycine/TBS to reduce the nonspecific recognition by the second set of antibodies. Incubation with the second set of antibodies was the same as the first set of antibodies. Finally, the coverslips were mounted in mounting buffer (100 mM n -propyl gallate, 50% glycerol in PBS, pH 7.4) and the samples were viewed with a Nikon microphot-FXA or with a laser scanning confocal system (MRC 600; Bio-Rad Laboratories) coupled to a Zeiss Axiophot microscope through a 60× oil immersion objective. Images were processed using Photoshop software (Adobe Systems, Inc.) and photographed from the monitor screen. To understand the function of a predominant nonerythroid 4.1R isoform (135 kD), we detected its binding partners by using a yeast two-hybrid system. Using the fusion protein Gal4-BD/135 kD 4.1R as a bait in pAS2-1 and a human brain cDNA library fused to Gal4-AD in pACT2, we screened ∼8 × 10 6 transformants. 79 positive clones were obtained, out of which 35 clones contained sequences that when translated would correspond to either amino acid residues 1476–2115 or 1697–2115 of NuMA . When Gal4-BD/22–24 kD 4.1R was used as a bait , some of the positive clones obtained also corresponded to the residues 1697–2115 of NuMA . This interaction of 4.1R and NuMA in the two-hybrid assays seemed to be specific, because neither the Gal4-BD 4.1R hybrid interacted with the unfused Gal4-AD, nor did the Gal4-AD–NuMA hybrid (clones obtained from the library screening) interact with the unfused Gal4-BD (data not shown). Three NuMA isoforms of 230, 195, and 194 kD have been reported. These isoforms arise by alternative splicing from a common precursor of a single NuMA gene but differ at their COOH termini . The NuMA protein has three different domains . The NH 2 - and COOH-terminal's globular domains correspond to residues 1–207 and 1729–2115, respectively. The middle α-helical coiled-coil domain comprises residues 208–1728. The positive clones obtained in the two-hybrid assays corresponded to the COOH-terminal end of the coiled-coil domain and the COOH-terminal globular domain. This could have derived from the 230-kD NuMA isoform because a part of these sequences was not detected in 195-kD and 194-kD isoforms of NuMA. In the 230-kD NuMA isoform, residues 1972–2007 were shown to contain a nuclear localization signal; the residues 1538– 2115 are necessary and sufficient for its spindle association. This isoform of NuMA is present in interphase nuclei and has been shown to concentrate at the polar regions of the spindle apparatus in mitotic cells . To identify the domains of 4.1R and NuMA responsible for their interaction, we expressed different domains of 4.1R as fusion products of Gal4-BD in pAS2-1 and cotransformed into Y190 with different domains of NuMA expressed as Gal4-AD fusion products in pACTII or pACT2, as shown in Fig. 2 . The full-length (135-kD) 4.1R, its 80-kD isoform, and the 22–24-kD domain interacted with the COOH-terminal domain, but not the NH 2 -terminal or the coiled-coil domain of NuMA, in the yeast two-hybrid assays. The HP, 30-, 16-, or 10-kD domain of 4.1R did not interact with NuMA. None of the domains of 4.1R, when expressed as Gal4-BD fusion proteins in Y190, expressed the reporter genes by themselves or in combination with the Gal4-AD in pACT2 alone (data not shown). These data suggest that the COOH-terminal domains of 4.1R and NuMA are necessary and sufficient for their interaction. To delineate the minimal sequences of 4.1R and NuMA that interact, we examined the interactions between different segments of the COOH-terminal domains of both the 4.1R and NuMA in the yeast two-hybrid assays, by expressing segments of the COOH-terminal domain of 4.1R as Gal4-BD fusion proteins and that of NuMA as Gal4-AD fusion proteins, respectively. Neither the deletion of the amino acids encoded by the part of exon 17 that belongs to the 22–24-kD domain of 4.1R (exons 18–21/ pGBT9), nor the deletion of the amino acid residues encoded by exons 17, 18, and 19 (exons 20 and 21/pGBT9) affected the interaction between 4.1R and NuMA. Deletion of the peptide segment encoded by exon 21 (exons 18–20/pGBT9) abolished the interaction of 4.1R with NuMA, although this segment alone (exon 21/pGBT9) was not sufficient for binding to NuMA . The amino acids encoded by exons 20 and 21 of 4.1R (exons 20 and 21/pGBT9) were sufficient for its association with NuMA. However, the amino acids encoded by exon 20 alone (exon 20/pGBT9) did not bind to NuMA. Therefore, the extreme COOH-terminal 62 amino acids of 4.1R derived from exons 20 and 21 are required and sufficient for its interaction with NuMA. The two different clones of NuMA obtained from the yeast two-hybrid screening started at positions corresponding to amino acids 1476 and 1697 of NuMA. Peptide segments of NuMA representing amino acids 1476–1695 or 1697–1787 did not interact with 4.1R . Deletion of NuMA sequences representing the amino acids from the COOH-terminal end to 1804 resulted in a weak interaction with 4.1R, whereas amino acids 1788–1810 showed substantial binding. Therefore, we believe that amino acids 1788–1810 of NuMA are necessary and sufficient for its interaction with 4.1R. These results suggest that the interaction between 4.1R and NuMA occurs through the amino acids encoded by exons 20 and 21 of 4.1R, and the amino acids 1788–1810 of NuMA. To confirm the direct interaction between 4.1R and NuMA, the association was further analyzed by an in vitro binding assay. GST-tagged 135-, 80-kD, and different domains (HP, 30, 16, 10, and 22–24 kD) of 4.1R were expressed and purified . Because of the insolubility of the 30-kD peptide, there was minor host protein contamination in preparations of 30 kD/GST and 80 kD/GST fusion proteins (lanes 2 and 8, respectively). In 16 kD/GST preparation, another protein band, probably a degradation product, was also observed (lane 3). The authenticity of these fusion proteins was verified with the corresponding antibodies (data not shown). Immobilized GST-tagged 4.1R proteins were incubated with in vitro translated and [ 35 S]methionine-labeled NuMA peptides corresponding to amino acids 1697–2102 (NuMA1/TOPO), 1697–1889 (NuMA2/TOPO), or 1831–2102 (NuMA3/TOPO). Retention of NuMA peptides on the beads was analyzed by SDS-PAGE as described in Materials and Methods. The yeast two-hybrid assays showed that NuMA sequences required for its interaction with 4.1R are located within amino acid sequences 1788–1810. As shown in Fig. 4 , B and C, both NuMA1/TOPO and NuMA2/TOPO that contained these sequences bound strongly to both 135-kD and 80-kD 4.1R isoforms and its 22–24-kD domain. The HP, 30-, 16-, and 10-kD domains, or GST alone did not bind to NuMA. NuMA3/TOPO, which did not contain these sequences, did not bind to any of the 4.1R/GST fusion proteins . These results are consistent with that of the yeast two-hybrid assays, and further confirm the specific interaction between 4.1R and NuMA. We verified the expression of 4.1R and NuMA in HeLa and MDCK cells, both at the RNA and protein level. A 6.5-kb 4.1R mRNA and a 7.2-kb NuMA mRNA were detected in HeLa and MDCK cells (data not shown). Affinity-purified antibodies specific for HP (anti-HP 4.1) recognized four different protein bands in cytoplasmic protein fraction (molecular masses of ∼35, 45, 76, and 135 kD), two bands in the nuclear protein fraction (35 and 135 kD), and a major 135-kD band in the nuclear matrix fraction of MDCK cells . However, no protein band was seen when a replica blot was immunoblotted with the preimmune serum . To verify that the protein bands recognized by anti-HP 4.1 Ab are also recognized by other anti-4.1R antibodies, replica blots were also immunoblotted with antibodies specific for exon 19 and the 10-kD domain of 4.1R . In addition to other bands, both the anti-24 kD Ab and anti-10 kD Ab recognized a predominant 135-kD protein band in cytoplasmic, nuclear, and nuclear matrix fractions, suggesting that the 135-kD protein band recognized by the anti-HP 4.1 Ab is 4.1R. Unexpectedly, the anti-24 kD Ab did not show an 80-kD band in any of the fractions. This could have resulted from a low level of expression of 80 kD isoform(s) containing exon 19 in these cells. The anti-10 kD Ab revealed the presence of both the 135- and 80-kD 4.1R isoforms in the cytoplasmic and nuclear fractions, and additionally detected the presence of 4.1R epitopes in a predominant ∼110 kD polypeptide that was localized both in the cytoplasmic and nuclear fractions . Similar distribution of 4.1R was observed in MDCK cells using anti-HP 4.1 antibodies and immunofluorescent staining (data not shown). The 135-kD isoform of 4.1R was predominantly localized in the nucleus, centrosomes, and to some extent at the points of cell–cell contact of interphase cells. Also, it was localized in the mitotic spindle and spindle poles of dividing cells. These results are consistent with recent studies that 4.1R epitopes are localized in centrosomes, nucleus, and nuclear matrix . The anti-NuMA antibodies recognized two immunoreactive bands of ∼210 and 230 kD in the nuclear and nuclear matrix protein fractions, and a 210-kD band in the cytoplasmic fraction . This is consistent with the observation that two different isoforms of NuMA are expressed in interphase cells . Previously published results suggest that NuMA is localized in the nucleus of interphase cells. We believe that the minor fraction of NuMA seen in the cytoplasmic fraction occurred because of some cell nuclei lysis during cell fractionation. To assess the quality of subcellular fractionation and document that the 4.1R isoform(s) seen in the nuclear and nuclear matrix fractions are not due to contamination of cytoplasmic proteins, different subcellular fractions were also immunoblotted with antibodies to proteins known to localize in cell cytoplasm . Immunoblotting with anti–Na + -K + -ATPase α1 Ab and antidynein mAb detected an ∼120- and 70-kD band, respectively, only in the cytoplasmic fraction. This suggests that the 4.1R isoform(s) seen in the nuclear and nuclear matrix fractions is not the result of contamination of 4.1R isoforms localized in the cytoplasm. Because 4.1R isoforms are documented to associate with centrosomes at mitosis and even after microtubule depolymerization , different subcellular fractions were immunoblotted with antibodies for centrosomal markers such as γ-tubulin and pericentrin . This was done to abrogate the possibility that 4.1R isoforms seen in nuclear and nuclear matrix fractions could have occurred because of centrosomal contamination in these fractions. Immunoblotting of different subcellular fractions with anti–γ-tubulin Ab and antipericentrin Ab showed the corresponding 48-kD γ-tubulin and 220-kD pericentrin only in cytoplasmic fraction. This suggests undetectable centrosomal contamination in the nuclear and nuclear matrix fractions. To further localize the association of 4.1R and NuMA, we used double immunofluorescence staining and confocal microscopy, and analyzed MDCK cells at different stages of cell cycle (see Materials and Methods). In interphase, 4.1R epitopes were found diffusely in the cytoplasm, but predominantly inside the nucleus , whereas NuMA epitopes were found mainly inside the nuclei . In mitotic cells, both 4.1R and NuMA intensely stained the spindle poles as shown in B1/C1 and B2/C2, respectively. NuMA epitopes were mainly found in the nucleus of the newly forming daughter cells (D2), whereas 4.1R epitopes were seen in the cytoplasm as well as nuclei (D1). The yellow color produced by superimposing green and red suggested that some 4.1R and NuMA epitopes colocalized in interphase nuclei (A3), in the mitotic spindle and spindle poles (B3 and C3), and in the daughter cell nuclei (D3). Krauss et al. , using immunogold staining and electron microscopy, observed that in the interphase 4.1R epitopes were primarily found in the vicinity of dense bodies. Zeng et al. reported that NuMA was present in subsets of the core filaments. However, using double label immunofluorescent staining and confocal microscopy, Krauss et al. also observed 4.1R foci within the nuclear area that was stained by NuMA. These results, the diffuse distribution of 4.1R during interphase and its segregation at the spindle poles during mitosis, are similar to those of other proteins essential for mitotic spindle assembly such as, NuMA , dynein , HSET , and Eg5 . To determine whether the native 4.1R interacts with NuMA in vivo, coimmunoprecipitation assays were performed using nuclear extracts from MDCK cells. MDCK nuclear extracts were subjected to coimmunoprecipitation with anti-HP 4.1 or anti-NuMA antibodies. Coprecipitated polypeptides were detected by immunoblot staining using anti-HP 4.1 or anti-NuMA antibodies. As shown in Fig. 7 , A and B, when preimmune serum, mouse IgG, or anti-p53 mAb were used to immunoprecipitate MDCK nuclear extracts, neither 4.1R nor NuMA precipitated. However immunoprecipitation with anti-HP 4.1 Ab and subsequent detection with anti-NuMA mAb showed that anti-HP 4.1 Ab precipitates two proteins of ∼210 and 230 kD . These proteins comigrate with two polypeptides of similar molecular mass that were immunoprecipitated by anti-NuMA mAb and were detected by immunoblotting using anti-NuMA antibodies. Similarly, immunoprecipitation with anti-NuMA mAb precipitated a protein of ∼135 kD that comigrates with 4.1R and immunoreacts with anti-HP 4.1 Ab . These results suggest that a nuclear isoform of 4.1R and NuMA is contained in the same protein complex in vivo. The coprecipitation of NuMA by anti-HP 4.1 antibodies, detection of 4.1R that coprecipitated with NuMA by anti-HP 4.1 antibodies, and the molecular mass of 4.1R isoform that coprecipitates with NuMA (135 kD) suggest that the 4.1R isoform(s) that interact(s) with NuMA is a 135-kD isoform that contains HP observed in nonerythroid isoforms. To find out which fractions of 4.1R and NuMA associate together, the efficiencies of immunoprecipitation and coprecipitation were determined. Quantitation from Fig. 7 , A and B, shows that ∼29% of 4.1R was precipitated by anti-HP 4.1 Ab (that coprecipitated ∼8% NuMA) and ∼47% of NuMA was precipitated by anti-NuMA mAb which in turn brought down ∼15% 4.1R. The limited efficiency of the immunoprecipitation could have happened because of insufficient amounts of antibodies in the immunoprecipitation assays. The lower efficiency of coimmunoprecipitation as compared with that of the immunoprecipitation suggests that only a fraction of these molecules associate together in vivo. This is consistent with the subcellular fractionation and immunofluorescent staining results. To examine the possible interaction of other isoforms of 4.1R with NuMA, immunoprecipitates of anti-NuMA mAb were also analyzed by antibodies specific to other domains of 4.1R. Analysis of anti-NuMA mAb immunoprecipitates by immunoblotting using anti-24 kD Ab (data not shown because of the poor quality of the immunoblot) revealed the presence of the 135-kD 4.1R isoform that was detected by using anti-HP 4.1 Ab, but did not show the presence of other isoforms of 4.1R. These data further support our contention that a 135-kD isoform(s) of 4.1R interacts with NuMA, although it does not eliminate the possibility that other isoforms of 4.1R or 4.1-like genes also interact with NuMA. Because actin, spectrin, and calmodulin were reported to interact with 4.1R, immunoprecipitates of anti-HP 4.1 Ab and anti-NuMA mAb were also analyzed by Western blot using antibodies specific to these proteins. However, actin , spectrin , or calmodulin (data not shown) was not detected in the immunoprecipitates of anti-HP 4.1 Ab and anti-NuMA mAb. To test whether 4.1R might also reside in association with other components of the mitotic apparatus, we asked whether it could be found in association with other proteins that are known to organize the mitotic spindle and spindle poles. Increasing evidence from different laboratories suggests that NuMA, dynein, and dynactin form a complex that organizes the spindle pole and stabilizes the mitotic spindle . Therefore, we enriched cells with mitotic spindle and spindle pole organizing components by using double thymidine block and nocodazole. Afterwards, we performed immunoprecipitation in highly synchronized mitotic HeLa extracts using anti-HP 4.1 Ab, anti-NuMA mAb, antidynein (70.1) mAb, or antidynactin (p150 glued , the 150-kD component of the dynactin complex) mAb. We looked for the presence of 4.1R, NuMA, dynein, and dynactin in the immunoprecipitates by immunoblot. An irrelevant antibody (anti-p53 mAb) and mouse IgG were used as controls for mAbs. To rule out nonspecific aggregation as a basis for detecting proteins on immunoblots and in the immunoprecipitates, preimmune rabbit serum was used as a control for Abs. As shown in Fig. 8 A, ∼140-kD protein, immunoprecipitated and detected by immunoblot staining using anti-HP 4.1 Ab (lane 1), coprecipitated with NuMA, dynein, and dynactin (lanes 3, 5, and 6, respectively) when immunoprecipitation was performed with anti-NuMA mAb, antidynein mAb or antidynactin mAb (p150 glued ), but not by preimmune serum, anti-p53 mAb, or mouse IgG (lanes 2, 4, and 7, respectively). This ∼140 kD protein corresponds to a 135-kD 4.1R isoform. We previously have documented such an alteration in the mobility of 135-kD isoforms of 4.1R because of phosphorylation at mitosis . When these blots were stripped and reprobed with antibodies specific for the COOH-terminal 24-kD domain of 4.1R , the ∼140-kD protein was seen in the immunoprecipitates of anti-HP 4.1, anti-NuMA mAb, antidynein mAb, and antidynactin mAb (lanes 1, 3, 5, and 6, respectively). No other convincing protein band corresponding to other 4.1R isoforms was seen in the immunoprecipitates (data not shown). Thus, other isoforms of 4.1R are unlikely to associate with NuMA, dynein, or dynactin. When a replica of the blot in Fig. 8 A was probed with anti-NuMA mAb , an ∼230-kD band corresponding to NuMA was seen in the immunoprecipitates of anti-HP 4.1 Ab, anti-NuMA mAb, antidynein mAb, and antidynactin (p150 glued ) mAb (lanes 1, 3, 5, and 6, respectively), but not in any of the control antibodies (lanes 2, 4, and 7). Similar analysis with appropriate antibodies detected dynein and dynactin in immunoprecipitates of anti-HP 4.1 Ab, anti-NuMA mAb, antidynein mAb, and antidynactin (p150 glued ) mAb (lanes 1, 3, 5, and 6, respectively), but not in any of the control antibodies used for immunoprecipitation (lanes 2, 4, and 7). These results suggest that 4.1R, NuMA, dynein, and dynactin associate together in vivo, and nonspecific aggregation is unlikely to account for this association. We attempted to quantitate how much 4.1R associates with NuMA and vice versa, by analyzing the efficiencies of their immunoprecipitation and coprecipitation. Representative immunoblots of anti-HP 4.1 Ab and anti-NuMA mAb immunoprecipitated, along with the supernatant fractions probed with anti-HP 4.1 Ab and anti-NuMA mAb, are shown in Fig. 8 , F and G, respectively. Quantitation of the efficiencies of immunoprecipitation and coprecipitation from these blots showed that ∼39% of total cellular 135-kD 4.1R protein was immunoprecipitated by anti-HP 4.1 Ab which in turn brought down 11% of total cellular NuMA. Similarly, ∼31% of NuMA protein was immunoprecipitated by anti-NuMA mAb that brought down ∼10% of 4.1R. It appears that under these conditions about one-third of NuMA and 4.1R remain associated and antibodies, not the antigens, are the limiting factors. To document the presence of other candidate proteins in this protein complex, anti-HP 4.1 Ab immunoprecipitates were examined by gel electrophoresis and silver staining . Approximately 15 prominent protein bands of very high molecular mass (∼40 kD) could be seen on the gel. Protein bands that correspond to NuMA, the p150 glued subunit of the dynactin complex, 135-kD 4.1R, and dynein in immunoblots were seen on the gel and are indicated in Fig. 8 H. To obtain an estimate of the molar ratios between NuMA, 4.1R, dynein, and dynactin in the immunoprecipitated protein complex, we determined the relative amounts of these proteins by scanning a picture of the silver stained gel of anti-HP 4.1 Ab immunoprecipitates. The molar ratio between NuMA, 4.1R, and dynactin was found to be 1:1.3:1.4. The molar ratio of the dynein intermediate chain could not be determined because of higher background staining. However, different proteins may not stain equally by silver stain . To examine the possible participation of other proteins in this protein complex, known to bind to 4.1R such as actin, spectrin, and calmodulin, we analyzed the anti-HP 4.1 Ab and anti-NuMA mAb immunoprecipitates by immunoblot staining using antibodies specific for these proteins (data not shown). Neither actin, spectrin, nor calmodulin was detected in these immunoprecipitates. The identities of other proteins remain to be identified. Our results extend the previous findings by Merdes et al. and Gaglio et al. . Merdes et al. presented persuasive evidence of dynein and dynactin in a mitotic spindle complex with NuMA. Gaglio et al. also showed that microinjection of dynein-specific antibody blocked spindle formation by inhibiting the association of dynein with the mitotic spindle and dislocating NuMA from spindle poles. Our findings are consistent with these observations and suggest that 4.1R, at least a 135-kD isoform, also participates in this complex. Results obtained with control antibodies, coupled with the nondetection of actin, spectrin, and calmodulin, support the contention that our coimmunoprecipitation results are far more likely to reflect a specific association in vivo rather than a nonspecific aggregation artifact of the immunoprecipitation assays. Because the above results suggested that there may be a direct interaction between NuMA and 4.1R, and NuMA is a nuclear protein, and is known to play a role in the organization of mitotic spindle and spindle poles, we asked if overexpression of 4.1R could disturb the intracellular distribution of NuMA or disrupt the organization of mitotic spindle and spindle poles. We transiently transfected HeLa cells with 135 ++ kD/GFP construct and examined the effect of overexpression of this isoform on transfected cells. Different degrees of 135 ++ kD/GFP expression were observed in transfected cells 10 h after transfection. In most of the transfected cells, 135 ++ kD/GFP fusion proteins were located in the cytoplasm. However, in some cells the fusion proteins could be detected in both cytoplasm and nucleus. As shown in Fig. 9 , GFP proteins were expressed in both nuclei and cytoplasm of pEGFP-C1 vector transfected HeLa cells (A1), whereas NuMA was exclusively localized in the nuclei (A2). This suggests that the expression of GFP protein alone did not alter the localization of NuMA in the interphase nuclei. In contrast, nearly all of the cells that strongly expressed 135 ++ kD/GFP, regardless of whether the localization of 135 ++ kD/GFP was in the nucleus or cytoplasm, showed altered localization of NuMA in the interphase cells. NuMA was distributed diffusely in these cells . The dispersed localization of NuMA was not apparent in the cells that weakly expressed the fusion protein, as shown in the top cell of Fig. 9 B2. In the cells that expressed the GFP alone, the localization of NuMA appeared to be defined by a boundary. In cells that highly expressed the 135 ++ kD/GFP fusion protein, NuMA was diffusely distributed well beyond this boundary. As shown in Fig. 9 , B3 and C3, there was an enhancement of yellow in the cytoplasm due to a combination of green and red, indicating the colocalization of these two proteins. This possibly suggests the appearance of 4.1R and NuMA complex aggregates in the cytoplasm, where it is not found normally. Conversely, there was a loss of staining signal in the nucleus in Fig. 9 B2 as compared with nuclear staining of NuMA in vector/nontransfected cells. These results suggest that overexpression of 135 ++ kD 4.1R isoform in HeLa cells may disrupt the organization of nuclear structural proteins such as NuMA. Cells that strongly expressed 135 ++ kD/GFP also gradually disappeared from the population 3–4 d after transfection. Furthermore, the selected disappearance of cells that expressed the fusion protein raised the possibility that such disruption of the normal organization of NuMA could adversely affect the cell cycle progression since the majority of 135 ++ kD/GFP overexpressed cells were eliminated from the cell population. It is interesting to note that we have not been able to identify cells that strongly overexpress the 135 ++ kD/GFP fusion protein in mitosis, whereas cells that expressed the fusion protein very weakly have been detected in mitosis. This preliminary observation is consistent with the notion that overabundance of 135 ++ kD 4.1R could indeed disrupt mitosis. Previous studies, from our laboratory and others , have documented the existence of multiple isoforms of 4.1R. Many of these isoforms are present in nonerythroid cells; their function and biological significance remain unknown. In this study, we demonstrate an interaction between a 135-kD nonerythroid isoform of 4.1R and NuMA by using a yeast two-hybrid system, in vitro binding assays, coimmunoprecipitation, and immunocolocalization studies. We also show that a 135-kD 4.1R isoform resides in association with mitotic spindle pole organizing proteins such as NuMA, cytoplasmic dynein, and dynactin. Furthermore, overexpression of a 135-kD isoform alters the normal distribution of NuMA in interphase cells that gradually disappear from the cell population. Taken together, our results suggest that a 135-kD 4.1R isoform interacts with NuMA in the nucleus and nuclear matrix of interphase cells and in the mitotic spindle and spindle poles of dividing cells. While the results in this paper strongly indicate that one or more isoforms of protein 4.1R interact with NuMA, they do not precisely pinpoint any one of the many potential forms that can be generated by alternative splicing of the 4.1R pre-mRNA. We believe that the isoforms of 4.1R that interact with NuMA are in the 135-kD molecular mass class even though NuMA interacts with both the 135-kD and 80-kD 4.1R isoforms in both yeast two-hybrid assays and in vitro binding assays. Indeed, NuMA does not even interact directly with HP that distinguishes the two molecular mass classes. These findings are compatible because the artificially created proximity between binding sites on each molecule in in vitro assays can allow them to interact, even if compartmentalization of the molecules, their proximity, orientation, posttranslational modification, or the presence of modifying factors precluded their interaction in intact cells. Further studies with additional domain specific antibodies and RT-PCR methods will be needed to define the exact isoform(s) that interact(s) in vivo. Our studies are the first that specifically implicate protein 4.1R, as opposed to other members of the protein 4.1 family (4.1G, 4.1N, 4.1B), as a likely participant in mitosis. Previous studies from our group and from other laboratories have reported similar localization and redistribution of 4.1 in a cell cycle–dependent manner. Krauss et al. reported the presence of 4.1 in centrosomes. Additionally, using double label electron microscopy, they have shown the colocalization of epitopes for 4.1 and the centrosome-specific autoimmune serum, 5051. Lallena and Correas also reported the localization of 4.1R in the nucleus. However, a recent study showed that epitopes for antibodies used in the above studies could be found among other protein 4.1 family members. In this present study, antibodies specific to HP, which is unique to 4.1R, were used. Therefore, our results strongly suggest that 4.1R isoform(s) containing HP localize(s) in the nucleus, centrioles, spindle fibers, and other intranuclear structures. Differential localization of isoforms of other cytoskeletal proteins has been reported. For example, isoforms of band 3 with different 5′ ends localize either to peripheral membranes or to perinuclear regions . β-Spectrin isoforms with different COOH termini also exhibit different subcellular localization and binding partners . Since the localization and redistribution of 4.1R during cell division that we have described here are similar to that of NuMA , these are consistent with its interaction with NuMA, and imply a novel location and role for a cytoskeletal protein. NuMA is well known for its role in the formation and stabilization of the mitotic spindle . Accumulation of NuMA has been shown at the mitotic spindle poles and at or near the minus ends of microtubules . Sequestration of endogenous NuMA by microinjection of anti-NuMA antibodies into mitotic cultured cells was shown to disrupt the bipolar mitotic spindles . The spindle association activity of NuMA was mapped to its COOH-terminal part . A recent study showed that the critical sequences for spindle pole localization are contained within the amino acid residues 1750–1800 of NuMA . However, the mechanism by which NuMA binds to microtubules is not clear. NuMA does not contain a microtubule-binding region of kinesin , Map2 , or tau , but has been shown to directly bind to tubulin and organize microtubule formation through the distal portion of its COOH-terminal domain . Because 4.1R does not contain a microtubule-binding motif, it is possible that the localization of 4.1R at the mitotic spindle and spindle poles occurs through its interaction with NuMA or other proteins that directly bind to microtubules. Two other proteins, dynein and dynactin, with whom NuMA has been shown to form a complex at mitosis , also possess microtubule binding domains and have been shown to organize and stabilize mitotic spindle and spindle poles . In a recent study, microinjection of dynein-specific antibodies into intact cells or immunodepletion of dynein from mitotic extracts disturbed assembly of mitotic spindle or mitotic aster, respectively, and prevented accumulation of NuMA at the spindle poles , suggesting that the accumulation of NuMA at the spindle poles may be dynein-dependent. However, the precise mechanism of organization of the microtubules or the localization of NuMA at the polar ends of the mitotic spindle is not clear. Several lines of evidence support individual roles for NuMA, dynein, and dynactin. In an immunodepletion experiment using frog extracts, Merdes et al. observed a complex between NuMA, cytoplasmic dynein, and dynactin; the depleted extract failed to assemble normal mitotic spindles. Although this observation suggests that the complex between NuMA, cytoplasmic dynein, and dynactin may have a role in the assembly and stabilization of mitotic spindle, NuMA was found to associate with microtubules independent of cytoplasmic dynein . Additionally, organization of microtubules to astral arrays and association of NuMA and dynactin with the microtubules of the astral arrays have been observed in mitotic HeLa extracts from which cytoplasmic dynein (a minus end–directed motor protein) and Eg5 (a plus end–directed motor protein) were depleted. This suggests that NuMA may be associated with a second minus end–directed motor protein yet to be defined . This view is further supported by the observation that NuMA accumulated at spindle ends in the absence of dynein activity and that inhibition of NuMA blocked the movement of microtubule seeds on spindle arrays . Because NuMA has the ability to form filamentous networks, it has also been suggested that it may be mechanistically involved in the organization of microtubules of the mitotic spindle and/or serve to counterbalance the forces exerted against the microtubules by the putative motor to which it is attached . The focus of the present study was to gain clues about the function of the novel 135-kD nonerythroid isoform of 4.1R by identifying its binding partners. We did not detect the presence of actin, spectrin, or calmodulin in the immunoprecipitates of nuclear or mitotic extracts, although these proteins are known to bind to 4.1R in erythrocytes. It is possible that in association with 4.1R isoforms, these proteins serve different functions. Their association with nuclear 4.1 isoform(s), like the 135-kD 4.1R, has not been documented. Nevertheless, it appears that a nuclear isoform of 4.1R interacts with NuMA in the interphase nucleus. It is not yet known if 4.1R and NuMA remain associated throughout the cell cycle or if their association is dynamic. The biological significance of this interaction is also not known at this time. However, the data shown in Fig. 9 suggest that perturbations of protein 4.1R alter the localization of NuMA. Cells that overexpress the 135-kD 4.1R isoform perished within a time frame of three to four cell divisions. Even though our data do not define a particular role for 4.1R, it is tempting to speculate that 4.1R might stabilize the interaction among NuMA, dynein, dynactin, and microtubules, in a manner analogous to its role in stabilizing the association of spectrin, actin, and integral membrane proteins in red cells. This is further supported by the demonstration that 4.1 epitopes are localized in nuclei, and asters and mitotic spindles. Moreover, the fact that antibodies specific for 4.1 perturb the formation of these structures in the Xenopus system lends additional support. However, more attempts to modify mitosis or in vitro reconstruction of astral arrays, by altering 4.1R expression or supply, will be required to test this notion. The binding sites between 4.1R and NuMA have been mapped to the amino acids encoded by exons 20 and 21 of the COOH-terminal domain of 4.1R and to amino acids 1788–1810 of NuMA . Because these residues of 4.1R are highly conserved among a growing 4.1R-like gene family it is possible that the polypeptide interacting with NuMA is a member of the 4.1R-like gene family. However, as discussed earlier, our results strongly implicate a 135-kD isoform of protein 4.1R. Of the three alternative COOH termini of NuMA that have been detected , amino acid residues 1788–1810 of NuMA, which are necessary for its interaction with 4.1R, are located within an alternatively spliced exon that is also known to code for the COOH terminus required for its nuclear localization and spindle association. A comparison of human and frog amino acid residues of NuMA in the region required for its interaction with 4.1R shows that this sequence is highly conserved (95%), although the proteins have only 48% overall homology . The especially strong conservation of the interacting sequences of NuMA and 4.1R could reflect an important role in a fundamental cell process, such as cell division. Thus, it appears that the isoform of NuMA that localizes to the nucleus and associates with spindle also binds with 4.1R, and further supports our earlier contention that 4.1R is present in the nucleus . The precise role played by 4.1R in spindle assembly is not known at present. However, our data strongly suggest that 4.1R is involved in some fashion. A role for 4.1R in cell division is supported by data that meet the same criteria used to document the initial involvement of NuMA, dynein, and dynactin in this process. Since 4.1R forms a complex with all these proteins, and can alter the localization of NuMA when perturbed, our data suggest that 4.1R may also be crucial for the organization of the mitotic spindle. Further work will be needed to establish the steps in these processes for which protein 4.1R is important. | Study | biomedical | en | 0.999996 |
10189367 | Purified Escherichia coli –derived core particles, as characterized by Crowther et al. ( 7 ), were obtained from Drs. Galina Borisova and Paul Pumpens (Biomedical Research and Study Centre, University of Latvia, Riga, Latvia). The particles were subjected to permeabilization and phosphorylation as described previously ( 31 ). Because of the RNA content of the particles, the incubation temperature and time for phosphorylation were increased to 37°C and 30 min, respectively. After reconstitution of particle integrity, entire core particles were separated from free nucleotides, protein kinases, and unassembled core proteins by sedimentation through a 0.5 ml 25% (wt/vol) sucrose cushion in TNE buffer (40 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) using a rotor (TL100; Beckman Instruments ) at 100,000 rpm at 15°C for 1 h. The pellet was resuspended in 50 μl TNE, aliquoted, and stored at −80°C until further use. The core particle concentration was determined using a core ELISA (>20 ng/ μl; ref. 47) using a standard of E . coli– expressed core particles (Chiron). 20 ng of phosphorylated or unphosphorylated core particles was separated on a 1% agarose gel, using agarose gel loading buffer without SDS. To disintegrate the particle structure, cores in parallel samples were incubated in 0.1% trifluoroacetic acid (TFA), heated to 50°C for 10 min, neutralized by adding 0.5 vol of 0.1 M Tris-HCl, pH 8.8, before loading on the gel. Proteins were blotted on an Immobilon-P membrane overnight ( Millipore ; 50). The membrane was blocked for 1 h at room temperature in 5% (wt/ vol) fat-free milk in PBS followed by addition of the first antibody (anti-HBc; DAKO) at a dilution of 1:4,000 in 5% milk/PBS for 2 h at room temperature. After washing (3× for 10 min in 0.1% milk/0.1% Tween 20/PBS at room temperature), the second antibody reaction was performed with HRP-labeled donkey anti–rabbit antibody (Dianova Co.) at a dilution of 1:5,000 in 5% milk/PBS for 1 h at room temperature. After washing, the antibodies were visualized by an enhanced chemiluminescence kit ( Boehringer Mannheim GmbH ). For analyzing the radioactive phosphorylation with [γ- 32 P]ATP ( Amersham ), the membrane was washed three times for 10 min in PBS before analysis by phosphoimaging . For quantification of core phosphorylation, 2 μl of the samples was spotted on GF/C filters (Whatman), air dried, washed twice for 10 min in ice-cold 10% (wt/vol) TCA and twice for 5 min in ice-cold 5% TCA. The filters were dried and protein-bound radioactivity was determined by liquid scintillation counting. For isopycnic centrifugation a CsCl gradient was performed with a density of 1.51–1.22 g/ml PBS in an SW60 rotor (Beckman). The 32 P-labeled core particle preparation was loaded on top and centrifuged at 35,000 rpm for 16 h at 10°C. The gradient was harvested in 20 fractions of 175 μl. 20 μl of each aliquot was subjected to core ELISA and to liquid scintillation counting. To analyze the dephosphorylation of phosphorylated core particles, 20 ng of particles was incubated with bacterial alkaline phosphatase (200 U/ml; New England Biolabs ) or with 4,000 U/ml calf intestinal phosphatase ( Boehringer Mannheim GmbH ) in 100 mM Tris-HCl, pH 7.5, 150 mM NaCl or 100 mM Tris-HCl, pH 8.5, 150 mM NaCl, respectively. In the control reaction, phosphorylated core particles were disintegrated by TFA, as described above, before dephosphorylation. Samples were incubated for 30 min at 37°C. Samples containing core particles and the unphosphorylated control were separated on an agarose gel, blotted, and exposed as described above. Disintegrated core protein samples were separated on a 16% SDS-PAGE, stained with Coomassie brilliant blue, and dried before autoradiography. HepG2.2.15 cells were grown on 16-cm dishes in 5% CO 2 at 37°C. After reaching confluency DMEM containing 3% FCS was added for 4 d. 500 ml of medium was collected and insoluble components were sedimented by centrifugation at 3,500 g for 30 min at 4°C. HBV of the supernatant was sedimented through 3-ml cushions of 25% (wt/wt) sucrose/TNE in an SW27 rotor at 27,000 rpm for 36 h at 4°C. Each pellet was resuspended in 150 μl TNE adjusted to a CaCl 2 concentration of 5 mM and incubated with 25 U/ml S7 nuclease ( Boehringer Mannheim GmbH ) for 1 h at 37°C. To remove the surface proteins from the virus, NP-40 was added to 0.1%. After incubation, insoluble components were sedimented through a 0.5-ml cushion of 25% (wt/wt) sucrose/TNE for 10 min at 10°C in a TL100 rotor ( Beckman Instruments ) at 100,000 rpm. The upper phase, including the interphase, contained the core particles. They were sedimented through a 0.5-ml cushion of 25% (wt/wt) sucrose/TNE for 2 h at 10°C in a TL100 rotor (Beckman) at 100,000 rpm. The pellet was resuspended in 200 μl TNE. FITC-BSA ( Sigma Chemical Co. ) was conjugated with NLS according to Görlich et al. ( 18 ). The NLSs used were the SV-40TAg NLS (PKKKRKVED; 18) and M9 domain of hnRNP (YNNQSSNFGPMK). Both contain a spacer (amino acid sequence CGGG; 18) at their NH 2 terminus. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Dietmar Linder and Monica Linder, Institute of Biochemistry, Department of Medicine, Giessen, Germany) of the conjugates showed, on average, 19 NLS were linked to 1 BSA molecule. Cells were grown in DMEM with 7% FCS on collagen ( Sigma Chemical Co. ) or Cell-Tak ( Becton Dickinson ) coated 12-mm coverslips in 24-well dishes in 5% CO 2 at 37°C. The cells were washed with 1 ml serum-free DMEM and permeabilized with 80 μg/ml digitonin ( Sigma Chemical Co. ) in DMEM for 10 min at 37°C ( 1 ). They were washed twice for 10 min at 4°C in washing buffer (transport buffer: 2 mM Mg-acetate, 20 mM Hepes, pH 7.3, 110 mM K-acetate, 1 mM EGTA, 5 mM Na-acetate/1 mM DTT) containing 1% BSA and 10% goat serum (Dianova) followed by a 10-min incubation at 37°C in a humidified box using washing buffer. Preincubation of permeabilized cells with 50 μg/ml WGA/ml transport buffer ( Boehringer Mannheim GmbH ) or 200 μg antibodies (anti-NPC antibody, mAb414 [BAbCO]/ml transport buffer, anti-NS2, or anticalnexin), was performed during this step. Permeabilized cells were incubated with 5 μg/ml core particles in 20 μl transport buffer containing 30 mg/ml rabbit reticulocyte lysate (RRL; Promega Corp. ); 10 μg/ml aprotinin ( Sigma Chemical Co. ), 10 μg/ml leupeptin ( Sigma Chemical Co. ), 10 μg/ml pepstatin ( Sigma Chemical Co. ) for 20 min at 37°C. If required, an ATP-generating system (1 mM ATP, 5 mM creatine phosphate, and 20 U/ml creatine phosphokinase; Sigma Chemical Co. ) or ATP-depleting system (7 mM glucose, 1 U/ml hexokinase; Sigma Chemical Co. ) was added ( 1 ). In some experiments, RRL was preincubated with peptides, representing parts of the COOH terminus of the core protein (amino acids 142–155, TLPETTVVRRRDRG; amino acids 158–168, PRRRTPSPRRR; amino acids 165–175, PRRRRSQSPRR; amino acids 173–183, PRRRRSQSRES), or the NLS of SV-40 T antigen (STPPKKKRKRKV), or lamin B2 (RSSRGKRRRIE) at a concentration of 2 mM for 10 min on ice. The peptides were synthesized by Dr. Ursula Friedrich (Institute of Medical Virology, Giessen, Germany) on an Applied Biosystems 431A Peptide Synthesizer, purified by HPLC, and analyzed by MALDI mass spectrometry (Dietmar Linder and Monika Linder). For incubation of cores in the absence of cytosolic proteins, BSA was added instead of reticulocyte lysate to the same protein concentration (30 mg/ml). In some experiments, importin (karyopherin) α (Rch 1) and β (gift of D. Görlich, ZMBH, Heidelberg, Germany) were added to BSA-containing buffer at a concentration of 30 ng/μl ( 16 ) each. For the transport assay, FITC-labeled BSA conjugate with SV-40TAg NLS was added to a final concentration of 20 μg/ml. After incubating for 20 min at 37°C, cells were washed in washing buffer as described above. When the phosphorylated E. coli– derived core (P-rHBc) was used for blocking the import of the FITC-BSA–M9 conjugate, P-rHBc was added at a concentration of 100 ng/μl to the assay in the presence of RRL and incubated for 20 min at 37°C as described above. In the control experiment, cells were treated identically but without addition of P-rHBc. After the binding reaction, cells were washed three times in washing buffer for 10 min at 0°C. The coverslips were loaded on a new reaction mixture containing reticulocyte lysate, ATP-generating system and 20 ng/μl of the FITC-BSA–M9 conjugate. After a second incubation period of 20 min at 37°C, the coverslips were washed as described above. To quantitate the amount of bound core particles, cells were scraped off the coverslip using a rubber policeman, counted in a Neubauer chamber, and lysed by addition of 100 μl 10 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 10 mM MgCl 2 , 150 mM NaCl containing 20 U DNase I, and 2 mg/ ml RNase A. Cells were incubated for 15 min at 37°C and lysis was controlled microscopically before analysis by ELISA. For immunofluorescence microscopy, cells were fixed onto the coverslips using 3% paraformaldehyde (Merck) in PBS, pH 7.4, for 30 min at room temperature. The coverslips were washed once in PBS and in PBS/0.1% Triton X-100 for 5 min. After two additional washings for 5 min in PBS at room temperature, cells were exposed to a mixture of anti-HBc antibody (1:200) and anti-NPC (1:1,000) in PBS/10% goat serum/1% BSA for 90 min at 37°C. Coverslips were washed in PBS three times for 5 min at room temperature and incubated with secondary antibodies (affinity-purified FITC-conjugated goat anti–rabbit IgG, H+L; Dianova; affinity-purified Texas red–conjugated goat anti–mouse IgG, H+L; Dianova) in PBS/10% goat serum/1% BSA for 40 min at 37°C. Cells were washed as described above and embedded in 50 mg/ml DABCO/moviol ( Sigma Chemical Co. and Hoechst, respectively). Microscopy was performed on a Zeiss fluorescence microscope, equipped with a 63× plan apochromat objective ( Zeiss ). Photographs were taken on Kodak TMAX 400 pro film. Negatives were developed at 1600 ASA. Confocal immunofluorescence microscopy was performed on a Leica DM IRBE microscope. Analysis of core-NPC colocalization and of the nuclear localization of FITC-labeled conjugates was done by using the TRITC- and FITC-fittings at a pinhole size of 0. 7 × 10 6 sheep anti–rabbit conjugated biomagnetic beads ( Dynal ) were washed twice in PBS according to the vendor's manual, resuspended in 500 μl PBS containing 2 μl anti-core antibody-containing rabbit serum (DAKO), and incubated overnight at 4°C. After washing as described above, 25 ng core was added and incubated overnight at 4°C. The beads were washed three times in 500 μl PBS, resuspended in 500 μl 0.1% BSA/ PBS, and incubated for 1 h at 4°C. Afterwards, the beads were added to 100 μl of RRL for 30 min at 37°C. In a control experiment, 2 mM of lamin B2 NLS (RSSRGKRRRIE) was added to the lysate. Further controls were performed by replacing lysate either with 300 ng importin β and 450 ng importin α in transport buffer, or by 300 ng importin β alone. The beads were washed three times in 500 μl PBS, 1× 500 μl PBS/0.1% Tween 20, and transferred to a new tube. After three cycles of washing in 500 μl PBS, the pellet was resuspended in 30 μl Laemmli buffer (66 mM Tris-HCl, pH 6.8, 10% [vol/vol] glycerin, 5% [vol/vol] β-mercaptoethanol, 2% [wt/vol] SDS, 0.015% [wt/vol] bromphenol blue), and denatured for 5 min at 96°C. The samples were separated on an 12% SDS-PAGE and blotted overnight to an Immobilon-P membrane ( Millipore ) by wet transfer. The membrane was blocked for 1 h at room temperature in 5% fat-free milk in PBS and the first antibody (anti-importin β, gift from D. Görlich) was added at a dilution of 1:4,000 in 5% milk/PBS for 2 h at room temperature. Washing, incubation with the second antibody, and detection were performed as described above. The in vitro generated cores (rHBc) used in this study were obtained by expressing the core protein in E . coli in the absence of other viral proteins. In the bacterial cytosol, the core proteins assemble into particles ( 43 ) that have the same size and symmetry as authentic cores ( 33 ). Instead of viral DNA, they contain unspecific E . coli RNA ( 3 , 13 , 39 ). When analyzed by isopycnic centrifugation in CsCl, the cores produced banded at 1.355 g/ml in CsCl , the same density as authentic cores (1.355–1.36 g/ml; ref. 22). In contrast to cores expressed in eukaryotic cells, E . coli– derived cores are not phosphorylated. To generate P-rHBc gradient purified cores were permeabilized using low salt, incubated in the presence of PKC and ATP, reconstituted in physiological salt solution, and repurified by gradient centrifugation ( 31 ). Using [ 32 P]ATP for labeling, the extent of phosphorylation was ∼12–24 phosphate groups per particle. In native agarose gel electrophoresis, both P-rHBc and rHBc comigrated as a homogeneous band . Alkaline phosphatase did not remove the phosphorus-32 label of intact core particles but after disruption of the particle structure . Thus, it was apparent that the phosphorylation sites were inaccessible to enzymatic hydrolysis as in authentic cores ( 15 ). The phosphorylation sites are located close to the COOH terminus of the core protein overlapping with an arginine-rich region ( 15 , 37 ) containing several putative NLS ( 59 ). Immunoprecipitation of core protein after disruption of particle structure using an antibody specific to a phosphorylated peptide derived from amino acids 166–177 demonstrated that serine 172 was phosphorylated by PKC (data not shown). Identical results were obtained for core proteins derived from human liver ( 37 ) or HBV DNA-transfected hepatoma cell lines ( 36 ). In addition, infectious HBV particles ( 48 ) from the stable HBV DNA-transfected hepatoma cell line HepG2.2.15 were purified from the culture medium. In contrast to in vitro generated cores, these cores are from mature viruses and contain the full complement of viral DNA, polymerase, and host cell components. Treatment of the virus with the detergent NP-40 released the cores from the surface proteins (P-hHBc; 24). These cores migrated in CsCl gradients with a density of 1.36 g/ml and did not show any reactivity to antisurface glycoprotein antibodies in ELISA or immunoblot after separation on a native agarose gel (data not shown). Thus, regardless of the source of the cores, P-rHBc and P-hHBc behaved similarly by several biochemical and immunological criteria. To determine whether the viral cores can associate with nuclei, we used the human hepatoma cell line, HuH-7, grown on coverslips. The plasma membrane was permeabilized using digitonin ( 1 ), the cytosol was washed out, and the E . coli– generated HBV cores (rHBc or P-rHBc) were added in the presence or absence of RRL, which is a source of cytosolic factors. In some samples, the lysates were complemented with either an ATP generating (+ATP) or depleting system (−ATP). After fixation, cores and NPCs were localized by double indirect immunofluorescence microscopy using core specific antibodies and antibodies to NPC. Immunoblotting of cores electrophoresed in agarose gels and quantitative ELISA assays (not shown) confirmed that both rHBc and P-rHBc reacted equally well with the anti-core antibody. The antibodies were selective for intact cores, since they bound to heat-denatured particles with ∼100-fold lower efficiency. The P-rHBc gave a strong rimlike fluorescence pattern characteristic of ligand association with the nuclear envelope . The labeling colocalized extensively with the one for NPCs . In contrast, core particles that had not been phosphorylated (not shown) or had been mock phosphorylated in the absence of PKC (rHBc) did not bind to the permeabilized cells , to the nucleus, nor to other cellular structures. Thus, the lack of association of the rHBc with the nucleus cannot be explained by competition with nonnuclear structures. Additionally, a differential entry of the different types of cores into the permeabilized cells seemed unlikely since they looked identical in electron microscopy ( 31 ) and behaved identically in biochemical analysis . The binding of the P-rHBc to the nucleus indicated that phosphorylated core proteins were essential for nuclear association. P-rHBc bound to nuclei only if a cell lysate was present . Inclusion of ATP-generating or -depleting systems had no detectable effect . When the reticulocyte lysate was replaced by buffer containing BSA, no binding was observed . Taken together, these findings showed that HBV cores interacted with the nucleus of permeabilized cells, and that binding depended on core phosphorylation and cytosolic factors but not on metabolic energy. Identical results were obtained with the human hepatoma cell line HepG2 and with the mouse fibroblast cell line LTK (data not shown). Confocal immunofluorescence microscopy showed that the bound cores were localized at the nuclear envelope and not imported deeper into the nucleoplasm , at least not in a form detected by the antibodies used. The nuclei were import-competent as shown by the efficient uptake of FITC-labeled SV-40TAg NLS-linked BSA . Preincubation of the cells with WGA, a lectin that binds to N -acetylglucosamine containing proteins of the NPC ( 11 ), prevented detectable nuclear import of the substrate . Phosphorylation-dependent binding of cores to nuclei was quantitated by an ELISA (Table I ). As already mentioned, the ELISA detected both types of cores equally well . 17% of the P-rHBc were bound to permeabilized cells. This corresponds to ∼3,400 core particles per nucleus. Of the rHBc, only 4% associated with the permeabilized cells. If the cores were added to cells without prior digitonin permeabilization, a background association of <4% was observed. Analysis of the supernatants after binding verified the presence of intact core particles (data not shown). Thus, unbound core particles were neither disassembled nor degraded during the assay. To characterize the site of association, binding assays were performed in the presence of agents known to block binding of karyophilic proteins to NPCs. Preincubation with WGA prevented detectable binding of cores and also reduced the signal of the anti-NPC labeling . Binding was also blocked by pretreating the permeabilized cells with an excess of anti-NPC antibodies . Antibodies against irrelevant proteins, e.g., anti-NS2 or against the cytoplasmic tail of calnexin ( 20 ), an integral membrane protein of the ER (data not shown), had no effect. These observations strongly suggested that cores bound to components of the NPC. Note, in Fig. 2 , anti-NS2, no labeling for NPC was performed since the competing NS2-antibody was generated in the same species. To map NLS on the cores, various synthetic peptides were added to the RRL before incubation with cores and cells. Four of the peptides corresponded to positively charged sequences in the COOH-terminal domain of the core protein. They were chosen because they resemble classical nuclear localization sequences found in karyophilic proteins and because they are close to the core phosphorylation sites. They had the following positions within the core protein and amino acid sequences: 142–155 (TLPETTVVRRRDRG), 158–168 (PRRRTPSPRRR), 165–175 (PRRRRSQSPRR), and 173–183 (PRRRRSQSRES). As controls, two peptides corresponding to the known NLS sequences of SV-40 T antigen (STPPKKKRKV) and lamin B2 were used ( 27 ). As shown by immunofluorescence microscopy, four of the peptides blocked binding of P-rHBc to the NPCs. These were peptides 158–168 and 165–175, and both control peptides . The other two peptides 142–155 and 173–183 did not block binding , although peptide 173–183 differed only by two amino acids from peptide 165–175. The similarity between these two peptides with regard to their basic amino acids strongly argues against an unspecific blocking caused by their positive charges. Peptides 158–168 and 165–175 also inhibited nuclear import of SV-40TAg-NLS linked FITC-BSA . These results suggested that the binding of HBV cores to the NPCs was mediated by factors involved in the nuclear import of proteins containing classical NLS. Furthermore, the data implied that the COOH-terminal domain of the core protein contained sequences that served as a nuclear pore binding signal (NBS). To determine whether binding of cores was mediated by importins, the experiment was performed in the presence of a buffer complemented with recombinant importins α and β. No other cytosolic components were present. The added importins conferred full binding activity to the P-rHBc , indicating that core-NPC association was mediated by importins. To confirm that the binding of the cores was mediated by the classical nuclear import pathway that involves both importin α and β, the assay was performed using either importin α or β separately. Neither component alone could promote binding of P-rHBc to the NPCs. The requirement of both importins suggested that an interaction of importin α to the NBS signal on the core particle surface was required for binding of importin β. To confirm the binding of importin β, authentic core particles were purified from the supernatant of HepG2.2.15 cells. As P-rHBc and cores from human liver ( 37 ), these cores are phosphorylated at serine residues between amino acids 166 and 177, which is a prerequisite for secretion of enveloped virus ( 58 ). For our immunofluorescence experiments ∼10 10 particles were used, an amount which cannot be purified from HBV of culture medium (∼10 6 /ml). Therefore, the binding of P-hHBc to importins was studied by coimmunoprecipitation performed with smaller amounts of cores. P-hHBc immobilized to biomagnetic beads by the particle-specific anti-core antibody reaction that precipitated importin β from reticulocyte lysate . Comparison with a standard dilution series of importin β showed that about four importin molecules were associated with one core particle. In vitro phosphorylated P-rHBc also coimmunoprecipitated importin β but not the unphosphorylated rHBc . To confirm that P-hHBc and HBV-derived cores do not interact with importin β directly without importin α, as shown for P-rHBc , a lamin B2 NLS that binds importin α was added to RRL. The loss of importin β precipitation by P-rHBc and HBV-derived cores showed the involvement of importin α in the importin β binding to these cores. To further confirm this finding, RRL was replaced by importin β containing buffer. The results documented in Fig. 7 , C and D (third lanes), showed that regardless of the origin or phosphorylation status of the core particles no coimmunoprecipitation of importin β with core occurred. In the control reaction, where importin α and β were added, importin β was precipitated by the P-rHBc and HBV-derived cores . The above data showed an importin-mediated binding of the core particles to the NPC. To test whether the binding of cores to the NPC occurred so close to the nuclear pore that nuclear import of other substrates was inhibited, nuclei of digitonin-permeabilized cells were pretreated with an excess of P-rHBc. Next, the cells were used in a transport assay with FITC-BSA linked to the M9-domain transport signal of hnRNPs. This substrate was chosen because its import does not require importin α and β involved in P-rHBc binding. A control without pretreatment of the cells with P-rHBc showed nuclear import of this substrate . In contrast, preincubation with P-rHBc abolished the nuclear import of the M9-FITC-BSA . The bound cores thus prevented uptake of an independently targeted karyophilic substrate. The target and transport of viral capsids to the nucleus constitutes a key step in the replication cycle of most DNA viruses and some RNA viruses. With the exception of influenza and retroviruses, little information exists about these processes ( 57 ). We studied the targeting of HBV cores by applying concepts and in vitro assays originally developed to analyze the cell biology of nuclear import of karyophilic cell proteins. These included the use of digitonin permeabilized cells and binding and uptake experiments with isolated ligands ( 16 , 40 ). We found that only phosphorylated HBV cores were specifically targeted to the nucleus by mechanisms and host factors involved in the NLS-mediated uptake of cellular proteins. Attachment occurred to NPCs and blocked the import of other karyophilic substrates, thereby giving evidence for association with the nuclear pore. The binding required the presence of importins α and β. The involvement of importins was confirmed by the coimmunoprecipitation of importin β from reticulocyte lysate using HBV-derived core particles. Thus, the contents of the authentic core particles, e.g., polymerase, hsp90, and viral DNA, had apparently no influence on nuclear binding. However, phosphorylation of the core subunits was necessary for exposure of NBS. The inhibition by specific peptides strongly implied that nuclear binding of HBV cores involved sequences present in the COOH-terminal portion of the core protein. During core assembly the pregenomic RNA, the viral polymerase ( 2 , 23 ), hsp90 ( 25 ), and PKC ( 32 ) are first encapsidated into a particle containing multiple core protein subunits. The COOH-terminal domain of the core protein is highly positively charged. In unphosphorylated cores, cryoelectronmicroscopy has shown that it is located inside the cores close to small holes in the capsid wall ( 60 ). It binds to nucleic acid ( 21 , 41 ) and also provides the phosphorylation site(s) for the protein kinase trapped in the central cavity ( 21 , 31 , 32 , 36 , 37 ). Since the phosphorylation sites on core proteins compete with the encapsidated RNA ( 31 ), phosphorylation of the core protein is most likely linked with DNA synthesis. Peptide inhibition studies suggested that the region exposed as an NBS may include the sequence between amino acids 158 and 175. As in authentic cores ( 15 , 37 ), the phosphate groups were not removed by alkaline phosphatase unless the particles were destroyed. Thus, the phosphates most likely remained hidden. This suggests that only the nonphosphorylated part of the COOH terminus was exposed on the particle surface. Many cases have been described in which phosphorylation either up- or downregulates nuclear transport of proteins. This is known for lamins ( 35 ), SV-40 T antigen ( 46 ), PKC ( 4 ), v-Jun ( 52 ), and NF-AT ( 49 ). The export of influenza viral RNPs also requires phosphorylation ( 56 ). Moreover, it has been suggested that phosphorylation of a small number of M proteins of HIV-1 by kinases trapped in the virion triggers M protein release from the viral membrane and exposure of NLSs close to the phosphorylation sites ( 12 ). Core proteins contain an NLS in their COOH terminus ( 10 ) overlapping with a nucleic acid binding domain ( 21 ) and serine phosphorylation sites ( 36 ). Unassembled core proteins, where this sequence is exposed, are imported into the nucleus of HBV-infected cells ( 36 ). Inside the nucleus, these imported core proteins spontaneously assemble into core particles ( 47 ) devoid of nucleic acids ( 19 ). Our results suggest that phosphorylation of core subunits induces a conformational change that exposes the COOH-terminal NLSs in such a way that the phosphoserine residues remain inaccessible to added phosphatases. The positively charged COOH-terminal domain may dissociate from the nucleic acids inside the core. They may protrude through the neighboring holes in the capsid wall and become partially exposed on the surface to serve as a nuclear targeting signal. This structural change may involve only a fraction of proteins in a core. In the P-rHBc used in this study, on average 12–24 of the 240 core proteins per core were phosphorylated. This was evidently sufficient to target the cores to the nucleus. Both importin α and β were necessary and sufficient for core binding to NPCs. Thus, HBV cores followed the classical nuclear targeting pathway and not alternative pathways used by protein A1 of U snRNPs ( 38 ), M9-like domains of hnRNPs ( 44 ), influenza virus nucleoprotein ( 55 ), and glycoconjugates ( 8 ). Importin α binds to positively charged, T antigen–like NLSs, whereas importin β is thought to bind to this complex and mediate attachment of the complex to the fibrils that extend from the NPCs into the cytosol. This binding reaction is energy independent (for review see 6, 17, 42). With a diameter of >25 nm, the core exceeds the size limit for NLS-coated colloidal gold particles that can enter through nuclear pores ( 9 ). Thus, import of DNA should involve disassembly or deformation of the core particle. We postulate that phosphorylation of core protein is an important control element in the viral life cycle. During encapsidation, the interaction with the COOH-terminal arginine-rich sequences of the core proteins helps to condense the pregenomic RNA into the small space offered by the central cavity . The binding of RNA prevents the encapsidated PKC from phosphorylating this core domain . During reverse transcription, the RNA is degraded. This allows the PKC to phosphorylate the COOH-terminal domain of some core protein subunits . Phosphorylation induces a conformational alteration in the core proteins. As a consequence, the COOH terminus is exposed on the particle surface that serves as a nuclear pore targeting signal. By association with importins α and β , the cores are targeted to the nuclear membrane and bind to the nuclear pores . There, they release their DNA/polymerase complex by unknown mechanisms , which is transported into the nucleoplasm . Alternatively, the viral cores can undergo budding at the intermediate compartment between the ER and the Golgi apparatus leading to virus formation . When the viral cores enter new host cells , they can again make use of the importins and the classical pathway of nuclear import. One of the potential advantages of this strategy is that as long as the viral DNA is packaged it can be moved through the cytosol to reach its defined targets. Therefore, the release of the bulky DNA molecule occurs only upon reaching the nuclear envelope. The molecular mechanisms of this pathway can now be elucidated experimentally. | Study | biomedical | en | 0.999999 |
10189368 | The HTLV envelope expression vectors used in this study are the previously described plasmids CMV-ENV-1 and CMV-ENV-2 , which contain the respective HTLV-1 and HTLV-2 sequences corresponding to the env , tax , and rex genes, under the control of the simian cytomegalovirus (CMV) promoter. The CMV-ENVΔPvuII and CMV-ENVΔPmaCI constructs were used as the respective negative controls . The mutated constructs coding for HTLV-1 glycoproteins with single amino acid substitutions were described elsewhere . The CMV-ENV438-stop plasmid encodes a soluble form of the HTLV-1 glycoprotein under the control of the CMV promoter . The human immunodeficiency virus type 1 (HIV-1) envelope expression plasmid pMA243, which is derived from an HIV-1 LAI provirus and has the capacity for encoding the viral proteins Env, Tat, Rev, and Vpu, was a gift from M. Alizon (INSERM U332, ICGM, Paris, France) . Oligonucleotide-directed mutagenesis of the sequence encoding the SU portion of the HTLV-2 envelope protein was performed as described elsewhere . The constructs coding for HTLV-1 envelope proteins with truncations or deletions were generated using standard cloning techniques. Positions in the amino acid sequence of the HTLV-1 envelope protein are numbered from the initiator methionine. The locations of truncations and deletions relative to other features of the HTLV-1 envelope protein are depicted in Fig. 6 . COS-1 and HeLa cells were obtained from the American Type Culture Collection. CosLTRLacZ cells, which are COS cells stably expressing the bacterial β-galactosidase gene ( lacZ ) under the control of the HIV-1 long terminal repeat (LTR), and HeLa-Tat cells, which are HeLa cells stably expressing the HIV-1 tat gene, were a gift from M. Alizon . Also provided by M. Alizon were the HeLa-P4 cells, which are HeLa cells stably expressing both the lacZ gene under the control of the HIV-1 LTR and the human CD4 cDNA . All cell lines were grown in DME containing 50 μg/ml gentamicin and 5% FCS, and supplemented with 300 μg/ml hygromycin B ( Calbiochem Corp. ) for the CosLTRLacZ cells, or with 500 μg/ml G-418 sulphate (Geneticin, Life Technologies, Inc.) for the HeLa-P4 cells. Cell cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere. The quantitative assays used to evaluate syncytium formation elicited by the HTLV-1, HTLV-2, or HIV-1 envelopes have been described elsewhere . In all these assays, the HIV-1 LTR-driven expression of β-galactosidase is transactivated by the Tat protein upon fusion of envelope-expressing cells with receptor-bearing indicator cells. To assess the dominant negative effect of the glycoprotein mutants on syncytium formation induced by the wt HTLV-1 or HTLV-2 glycoproteins, the mutated and wt envelope constructs (total amount: 3 μg DNA) were cotransfected into CosLTRLacZ cells seeded at 3 × 10 5 cells per 60-mm–diameter dish the previous day by a procedure using DEAE-dextran, chloroquine, and dimethyl sulfoxide . Immunofluorescence analysis of the cells cotransfected with the constructs for the wt HTLV-1 envelope and for a truncation mutant demonstrated that more than 95% of the cells that expressed one of the envelope constructs in fact expressed both of them (data not shown). Radioimmunoprecipitation analysis also assured us that, within the range of DNA quantities used in this study, the level of protein expressed correlated with the amount of DNA transfected. 1 d (HTLV-2) or 2 d (HTLV-1) after transfection, 5 × 10 5 HeLa-Tat cells were added as indicator cells. After a 24-h coculture, the amount of β-galactosidase was evaluated by a chemiluminescence assay for detection of the activity of this enzyme in cell lysates (Galacto-Light; Tropix) with a luminometer . To assess the effect of the HTLV-1 glycoprotein mutants on syncytium formation induced by the wt HIV-1 envelope, the mutated HTLV-1 envelope constructs (2.25 μg) and the pMA243 plasmid (5 ng) were cotransfected into COS-1 cells seeded at 3 × 10 5 cells per 60-mm–diameter dish the previous day . 2 d after transfection, 5 × 10 5 HeLa-P4 cells were added as indicator cells. After a 24-h coculture, syncytia were stained in situ and counted under a light microscope as described elsewhere . For each assay, the syncytium formation index gives the percentage fusion induced by the wt envelope in the presence of the glycoprotein mutant relative to that obtained in its absence. The envelope constructs were transfected into COS-1 or HeLa cells by the calcium phosphate precipitation method. 1 d after transfection, the cells were seeded onto glass slides (Lab-Tek; Nunc, Inc.) at 4 × 10 4 cells per 80-mm 2 well. The next day, cultures were rinsed in PBS and fixed with 4% paraformaldehyde for 15 min at room temperature, followed by quenching in 0.1 M glycine in PBS. Permeabilization and saturation were achieved by a 2-h incubation in PBS containing 0.05% saponin and 0.2% BSA, and all subsequent steps were performed in this buffer at room temperature. The cells were incubated for 90 min with the primary antibodies. These were mAb 1C11 (1:100; Epitope), which is a murine mAb directed to the HTLV-1 SU , and rabbit polyclonal antibodies directed to the α subunit of translocating chain-associating membrane protein (1:500, kindly provided by T.A. Rapoport, Department of Cell Biology, Harvard Medical School, Boston, MA) or to Rab1 (1:25, kindly provided by B. Goud, CNRS UMR 144, Institut Curie, Paris, France). Excess antibody was removed with five washes, and the secondary antibodies were allowed to bind for 60 min. These were FITC-conjugated goat anti– mouse IgG (1:400; Jackson ImmunoResearch Laboratories, Inc.) and cyanin 3–conjugated goat anti–rabbit IgG (1:300; Jackson ImmunoResearch Laboratories, Inc.). After five washes, the slides were mounted in Mowiol and observed with a confocal laser scanning microscope . The pinhole aperture was such that optical section thickness was 0.6 μm. “Bleed-through” from the FITC to the cyanin channel was negligible. Images were processed using the Laser Sharp software. Colocalization appeared as yellow pixels after merging sections recorded at the same z level in each channel. Immunoprecipitations of the HTLV-1 envelope glycoproteins were performed as described in our previous studies , using protein A–Sepharose CL-4B beads ( Pharmacia Biotechnology ) coated with a pool of sera from HTLV-1–infected individuals (provided by J. Coste, CRTS, Montpellier, France). For coimmunoprecipitation experiments, protein A–Sepharose beads were coated with rabbit anti–mouse Ig (Dako SA) plus purified 4D4 mAb, which is a murine mAb raised against a synthetic peptide covering the COOH-terminal domain (amino acids 287– 311) of the HTLV-1 SU (kindly donated by C. Desgranges and M.-P. Grange, INSERM U271, Lyon, France) . Immunoprecipitates were electrophoresed in SDS-13% polyacrylamide gels under reducing conditions (except where otherwise stated), and visualized by autoradiography. The envelope constructs were transfected into COS-1 cells (2.8 × 10 6 ) by the calcium phosphate precipitation method. 2 d after transfection, the cells were lysed in 0.8 ml of 100 mM Tris-HCl, pH 8.0, containing 100 mM NaCl, 1 mM CaCl 2 , and 250 mM n -octyl-β- d -glucopyranoside ( Sigma Chemical Co. ). The clarified lysates, together with 50 μl of size markers (kD): 66 BSA, 141 alcohol dehydrogenase, and 250 catalase, each at 9 mg/ml ( Sigma Chemical Co. ), were loaded onto continuous sucrose gradients (10 ml, 5–35% sucrose in 100 mM Tris-HCl, pH 8.0, containing 100 mM NaCl, 1 mM CaCl 2 , and 40 mM n -octyl-β- d -glucopyranoside) and centrifuged for 20 h at 4°C in an SW41 rotor at 38,000 rpm before fractionation from the bottom of the tube into 18 fractions of 600 μl. To analyze size markers, 60-μl aliquots were removed from the fractions and were separated by SDS-PAGE, followed by Coomassie blue staining. The remainders of the fractions were subjected to immunoprecipitation with a pool of sera from HTLV-1–infected individuals as described above. Immunoprecipitates were electrophoresed in SDS-10% polyacrylamide gels under reducing conditions, and proteins were transferred onto membranes (Immobilon-P; Millipore Corp. ). After saturation in PBS containing 0.05% Tween-20 and 5% skim milk, the membranes were incubated with a 1:3,000 dilution of the mAb 4D4 for 90 min at room temperature. Excess antibody was removed with six washes, and a 1:1,000 dilution of the second-step antibody (peroxidase-conjugated goat anti–mouse IgG; Jackson ImmunoResearch Laboratories, Inc.) was allowed to bind for 60 min. After eight washes, immunoreactive spots were detected by enhanced chemiluminescence ( Amersham Buchler GmbH ). We have previously described a series of HTLV-1 envelope glycoprotein mutants that are defective in cleavage of the precursor into the mature SU and TM products . The cleavage of the HTLV-1 glycoprotein precursor, which is required for envelope-mediated function, normally takes place in the Golgi apparatus . As for most integral membrane glycoproteins, transport into the Golgi apparatus is thought to be strictly dependent on proper folding and oligomeric assembly in the ER . We thus considered the hypothesis that our HTLV-1 glycoprotein mutants with defects in precursor cleavage might actually be defective in precursor assembly. We designed a dominant negative assay in which these cleavage-defective mutants were coexpressed with the wt HTLV-1 glycoprotein in transfected COS cells. In this approach, an assembly-competent mutant would be expected to dominantly interfere with wt envelope-mediated function by titrating the wt precursor in the formation of transport-defective heterocomplexes; in contrast, an assembly-defective mutant would spare the intracellular maturation of the wt glycoprotein and hence its function. Transfected cells expressing the wt HTLV-1 envelope at the cell surface are able to induce fusion with indicator cells expressing the receptor, leading to the formation of syncytia. Syncytium formation was thus chosen as a convenient assay to account for intracellular transport and hence function of the HTLV-1 envelope. Because several reports have suggested that the oligomerization domain of retroviral envelopes lies in the TM glycoprotein , we first investigated the abilities of HTLV-1 glycoproteins with mutations in the TM portion to interfere with wt envelope-mediated function. We have previously described eight single amino acid substitutions in the HTLV-1 TM that result in a lack of precursor cleavage and, consequently, in a complete loss of syncytium-forming ability (see Table I ). The mutated envelope expression plasmids were cotransfected with the wt envelope expression plasmid (CMV-ENV-1) at a ratio of 3:1, and syncytium formation was monitored. Each of the eight cleavage-defective TM mutants exerted a marked dominant negative effect over the wt envelope, manifested by inhibition of syncytium formation (Table I ). That all of the glycoproteins mutated in the TM portion were able to interfere in trans with wt envelope-mediated function prompted us to test for negative dominance of glycoproteins mutated in the SU portion. We have previously described 14 single amino acid substitutions distributed throughout the HTLV-1 SU that abolish precursor cleavage and hence envelope-mediated function (see Table II ). A marked dominant negative effect on the syncytium-forming activity of wt envelope was documented for 13 of these 14 SU mutants (Table II ). Thus, the vast majority of HTLV-1 envelope mutants with defects in cleavage of the precursor glycoprotein are trans-dominant negative. We examined whether the inhibitory effect exerted by the mutants was specific for the HTLV-1 envelope. For this purpose, we cotransfected each HTLV-1 glycoprotein mutant construct with an expression vector encoding the wt envelope glycoprotein of HIV-1 (pMA243), and we scored the number of syncytia elicited by the HIV-1 envelope. In contrast to their drastic effect on syncytium formation induced by the wt HTLV-1 envelope, the HTLV-1 glycoprotein mutants failed to interfere with syncytium formation induced by the wt HIV-1 envelope, even upon cotransfection of a 450-fold excess of the HTLV-1 plasmid (Tables I and II ). As a positive control, coexpression of CD4 with the HIV-1 glycoprotein gave the expected inhibition of syncytium formation . The dominant negative effect was thus specific, and could not be accounted for by some general alteration of the intracellular maturation or transport of membrane proteins. It has been shown that aggregation occurs in the ER of cells synthesizing misfolded proteins and is not restricted to products from a single polysome . It could thus be argued that the mutated glycoproteins might titrate the wt glycoprotein in the formation of large heteroaggregates rather than in a bona fide dimeric assembly process. To decide between these two hypotheses, we combined different approaches. We first examined the oligomeric structure of the envelope glycoproteins from transfected cell lysates by velocity sedimentation on sucrose gradients. Consistent with previous data , most of the wt HTLV-1 envelope precursor glycoprotein was recovered in a peak at the position expected for the dimeric form of this 61-kD protein . A small amount of faster-sedimenting material was also detected that might correspond to tetramer formation. A similar pattern of sedimentation was detected in lysates from cells coexpressing the wt glycoprotein plus a threefold excess of a cleavage-defective dominant negative mutant . Notably, the position of the envelope glycoproteins did not shift towards the bottom of the gradient, as would have been expected if aggregation had occurred. We also used nonreducing SDS-PAGE to analyze the envelope products radioimmunoprecipitated from lysates of cotransfected cells, because interchain disulfide-bonded complexes often arise in the ER of cells synthesizing misfolded proteins. No such complexes were detected (data not shown), further arguing against an aggregation bias. In addition to these biochemical approaches, we examined the extent of the trans-dominant negative interference exerted by the cleavage-defective mutants as a function of the mutant:wt ratio . In a cell that coexpresses the wt glycoprotein and an assembly-competent mutant, three dimer combinations are expected: homodimers of wt precursor subunits, homodimers of mutant precursor subunits, and heterodimers consisting of both subunit types. If dimerization is random, the distribution between these three combinations can be calculated for a variety of expression ratios . Cells were transfected with a constant amount of DNA containing various ratios of mutant to wt HTLV-1 envelope constructs (3:1, 1:1, and 1:3), and the resulting syncytium-forming activity was compared with that observed after transfection of the wt construct alone. As shown in Fig. 2 b for two TM and two SU mutants, the experimental indices of syncytium formation at each ratio approached the theoretical values for the corresponding proportions of wt homodimers expected from a random dimerization process. This corroborates our biochemical findings, since trapping of wt glycoprotein in a higher-order structure would have shifted the experimental curves to the right. Therefore, the trans-dominant negative effect most likely reflects random dimeric assembly of mutant and wt glycoproteins, with wt homodimers being the sole functional combination. The design of the dominant negative assay was based on the assumption that a cleavage-defective mutant with competence for assembly would prevent the wt glycoprotein from being transported to, and consequently cleaved in, the Golgi apparatus. We used two complementary approaches to ascertain that this was indeed the inhibitory step. First, we performed in situ immunofluorescence analyses to examine the intracellular localization of the wt HTLV-1 glycoprotein expressed in the absence or presence of a cleavage-defective mutant . When the wt envelope construct was transfected alone, most of the positive cells were committed to the formation of syncytia, and a granular staining scattered throughout the cytoplasm was detected . In contrast, the staining was confined to the perinuclear space in cells expressing any of the cleavage-defective mutants . We performed double-label immunofluorescence experiments to identify the mutant-retaining organelles. As shown in Fig. 4 , the staining for HTLV-1 glycoprotein mutant showed partial colocalization both with that for translocating chain-associating membrane protein , an ER marker, and with that for Rab1 , an intermediate compartment and cis-Golgi marker. The pattern of staining observed in cells coexpressing the wt glycoprotein and a cleavage-defective dominant negative mutant was similar to that in cells expressing the mutant alone . These results indicate intracellular retention of wt glycoprotein in the presence of a trans-dominant negative mutant. We next verified that the mutants that by themselves were defective in precursor cleavage also impaired in trans the cleavage of the wt envelope precursor . Indeed, when expressed in the absence of mutant, the wt precursor gave rise to its mature products as appreciated by detection of the 20-kD band corresponding to the TM glycoprotein , whereas in the presence of any of the cleavage-defective dominant negative mutants, the TM-gp20 was barely or not detected . Taken together, these data confirm our hypothesis that the dominant negative effect exerted by cleavage-defective mutants results from their ability to prevent it from being transported to and cleaved in the Golgi apparatus. Among the 22 cleavage-defective HTLV-1 glycoprotein mutants, the SU mutant Ser25-Arg represented a notable exception: it spared 72% of the syncytium formation elicited by the wt envelope (Table II ). Immunofluorescence microscopy showed that the Ser25-Arg mutant, like the other cleavage-defective mutants, was defective in intracellular transport . Upon its coexpression with wt glycoprotein, however, giant multinucleated cells with a granular staining scattered throughout the cytoplasm were detected . The Ser25-Arg mutant thus failed to abolish in trans the intracellular transport of the wt glycoprotein, in sharp contrast with the other cleavage-defective HTLV-1 glycoprotein mutants. Furthermore, we confirmed that the cleavage of the wt glycoprotein was only partially inhibited by coexpression of a threefold excess of the Ser25-Arg mutant . We also verified that the phenotype of the Ser25-Arg mutant was not due to a greater instability of this glycoprotein compared with the dominant negative mutants (data not shown). Thus, assembly of the HTLV-1 envelope precursor is impaired by a single amino acid substitution located at the NH 2 terminus of the glycoprotein . We asked whether HTLV-2, a human retrovirus phylogenetically related to HTLV-1, has comparable requirements for envelope precursor assembly. We mutated the serine residue at position 21 of the HTLV-2 envelope protein, equivalent to the serine at position 25 in HTLV-1 . Like the Ser25-Arg HTLV-1 mutant, the Ser21-Arg HTLV-2 mutant by itself exhibited a cleavage-defective phenotype (Table III ). It also failed to abolish wt envelope-mediated function when expressed in a threefold excess with the wt HTLV-2 glycoprotein, sparing 85% of the syncytium-forming activity (Table III ). This shows that, in both HTLVs, the substitution of one serine at the NH 2 terminus of the glycoprotein is sufficient to impair precursor assembly. We generated mutants with truncations or deletions to map the HTLV-1 glycoprotein domains involved in precursor assembly . We verified that none of them elicited syncytium formation when expressed alone (data not shown), and we tested them for negative dominance. A truncation mutant lacking the membrane-anchorage and intracytoplasmic domains (ENV438-stop) abolished the syncytium-forming activity of the wt HTLV-1 envelope . As for point mutants, the trans-dominant negative effect was specific, because the truncated HTLV-1 glycoprotein mutant did not interfere with the syncytium formation elicited by the wt HIV-1 envelope (data not shown). It also followed the titration curve expected from a random dimeric association of the two glycoproteins . The cleavage of the wt envelope precursor was greatly impaired in cells coexpressing the ENV438-stop mutant . Conversely, secretion of the soluble products (SU of normal size and truncated TM) of the ENV438-stop mutant was reduced in the supernatant of these cells (data not shown). We concluded that (a) the ectodomain of the HTLV-1 envelope protein contains structural information that is sufficient for precursor subunit association, and (b) the heterodimers formed by the full-length wt glycoprotein and a COOH-terminally truncated, soluble mutant are defective in intracellular transport even though both subunits by themselves are competent for this process . We further sought to determine which regions within the HTLV-1 glycoprotein ectodomain are critical for intracellular assembly. A major role could be attributed to the SU portion, because a truncated mutant lacking all the TM sequences (SU313-stop) was sufficient to exert a strong dominant negative effect . The phenotype remained unchanged after further truncations of the SU that removed its COOH-terminal third (SU201-stop) or even its COOH-terminal half (SU163-stop). Again, titration of wt glycoprotein followed the theoretical curve expected from a random dimerization process . The cleavage of the wt envelope precursor was also greatly impaired in cells coexpressing any of these three truncated proteins . In contrast, an SU glycoprotein deleted of its NH 2 -terminal half (SUΔ26-164) was less effective in trans-inhibition of wt function, sparing 45% of the syncytium formation . It was also less effective in trans-inhibition of wt glycoprotein cleavage . Therefore, the integrity of the NH 2 -terminal domain of the SU was both necessary and sufficient for full trans-dominant inhibition of the intracellular transport and hence the function of the wt envelope. These findings underscore the importance of the NH 2 -terminal domain in the intracellular assembly of the glycoprotein. We reasoned that removal of the SU might unmask a potential contribution of the TM portion in precursor assembly. Indeed, a deletion mutant lacking most of the SU sequences (TM284-488) exerted a partial dominant negative effect on wt function . The effect seemed mostly due to the COOH-terminal region of the ectodomain, and not to the leucine zipper-like domain, because an NH 2 -terminally deleted protein lacking the leucine zipper-like motif (TM384-488) still exerted a dominant negative effect, whereas a TM protein deleted of the COOH-terminal half of the ectodomain (TMΔ382-441) was ineffective in trans-inhibition . Thus, in addition to the contribution of the NH 2 -terminal domain of the SU, a role in intracellular assembly could be attributed to the COOH-terminal region of the TM ectodomain. Finally, we took advantage of truncation mutants to provide direct demonstration of the physical interaction between HTLV-1 envelope proteins having an intact NH 2 -terminal domain. We used the 4D4 mAb, which is directed to the COOH-terminal domain (amino acids 287–311) of the HTLV-1 SU , to perform coimmunoprecipitation experiments . The 4D4 mAb was able to immunoprecipitate similar amounts of precursor glycoproteins for the wt envelope, the Ser25-Arg mutant, or a trans-dominant mutant, Arg379-Gly , but did not allow immunoprecipitation of the COOH-terminally truncated envelope protein SU163-stop, which does not contain the 4D4 epitope . However, the 4D4 mAb did coimmunoprecipitate the SU163-stop protein together with the wt precursor glycoprotein in cells coexpressing the two proteins . Therefore, the dominant negative effect displayed by the SU163-stop mutant could indeed be accounted for by the physical association of the truncated protein with the wt precursor glycoprotein in the coexpressing cell. A comparable level of SU163-stop protein was also coimmunoprecipitated together with a trans-dominant glycoprotein, Arg379-Gly, confirming that competence for assembly resulted in coimmunoprecipitation . In contrast, a smaller amount of SU163-stop protein was brought down from cells coexpressing the Ser25-Arg mutated glycoprotein . Similar results were obtained when the SU201-stop mutant was tested instead of the SU163-stop mutant (data not shown). These data provide direct evidence that competence for assembly is impaired by a single amino acid substitution in the NH 2 -terminal domain of the glycoprotein. In this study, we designed a dominant negative assay to explore the basis for the transport defect of a series of mutants of the HTLV-1 envelope, a glycoprotein which is subject to a very tight quality control . To our surprise, we found that, provided the NH 2 terminus was intact, all mutated forms of the glycoprotein were capable of interfering in trans with the wt. Incompetence for assembly thus cannot explain the incompetence for transport of a large series of retained glycoproteins. It should be noted here that the mutated HTLV-1 glycoproteins were not retained in the ER alone, since we also observed partial colocalization with an intermediate compartment and cis-Golgi marker. This is reminiscent of the retention of a VSV G mutant, which has been shown to involve cycling between the ER, intermediate compartment, and cis-Golgi . It could be argued that, rather than resulting from an assembly process, the trans-dominant interference exerted by the HTLV-1 glycoprotein mutants might be due to a nonspecific effect, such as the titration of a factor required for the intracellular transport of membrane proteins. This is very unlikely, however, because the HTLV-1 glycoprotein mutants had no effect on another retroviral glycoprotein, that of HIV-1. Moreover, coimmunoprecipitation experiments provided direct demonstration of a physical interaction between mutated glycoproteins exerting a dominant negative effect and the wt HTLV-1 precursor glycoprotein. Another caveat with the dominant negative approach is that it could reflect the constitution of aggregates rather than a true competence for oligomeric assembly. Although this hypothesis seemed unlikely because of the specificity of the trans inhibition, we addressed it directly by showing that the HTLV-1 glycoproteins from cotransfected cells were not recovered as aggregates in sucrose gradients. Moreover, for all mutants tested, the experimental titration curves for the dominant negative effect followed the theoretical curve expected from a dimeric assembly of the wt and mutated glycoproteins. We infer from these observations that our dominant negative assay reveals a specific dimeric assembly process taking place between HTLV-1 envelope precursor glycoproteins in the living cell. For most membrane-anchored glycoproteins studied so far, assembly in the ER is a late step which takes place after folding of the monomeric subunits . We therefore expected that the majority of the transport-defective HTLV-1 glycoprotein mutants would be non– trans dominant, due either to a defect in the assembly step per se or to a defect in a previous folding step necessary for assembly. We found, on the contrary, that incompetence for assembly was rarely responsible for the intracellular retention of the mutated glycoproteins: 21 of the 22 glycoproteins incapable of transport were nevertheless capable of dimeric association with the wt precursor. These data suggest that the assembly process unraveled here is unlikely to occur as a late event in the acquisition of transport competence. The apparent discrepancy between our results and the previous studies reviewed in Doms et al. can be resolved if one considers the methodology employed in each case. In the kinetic analyses that suggested that the essentially complete folding of monomers is a prerequisite to oligomerization, velocity gradient sedimentation was generally used as the assembly assay. Such a technique can only pick those oligomers that are stable enough to withstand detergent solubilization and centrifugation . Alternatively, monoclonal antibodies have been employed, but they too may detect only the mature, stable oligomer. An assembly assay based on negative dominance is complementary to these biochemical methods, because it evaluates the functional consequences of an interaction having occurred between subunits within the cell, but which would not necessarily have withstood the experimental manipulations involved in protein isolation and may not necessarily correspond to the ultimate oligomeric conformation. With this in mind, our results suggest the occurrence of an early and transient assembly step of the glycoprotein, preceding the appearance of the stable oligomer having the quaternary structure required for transport. Studies with HA have also suggested that the intracellular assembly of membrane-anchored proteins is in fact a multistep process, although it is detected as a discrete event when studied by a single method. Even though the association of HTLV-1 envelope precursor subunits is presumed here to occur early in the maturation process, it is a posttranslational event. Indeed, our titration curves were consistent with a random association of monomers having arisen from different polysomes. This feature is shared by the HA glycoprotein , and is likely to be a general rule of the assembly of membrane proteins, because their confinement to the ER compartment and their oriented state in the plane of the membrane facilitate spatial proximity between subunits . By contrast, a posttranslational mechanism is unlikely to be adopted by cytosolic oligomeric proteins . Our work allowed us to map the structural domain involved in the ER association of HTLV-1 envelope precursor subunits to the NH 2 terminus of the glycoprotein. This conclusion was drawn from a number of concordant observations. First, of the 22 point mutants tested, the only one that spared the intracellular transport of the wt precursor had a single amino acid substitution at the NH 2 terminus of the glycoprotein. Second, this mutant did not allow coimmunoprecipitation of an assembly-competent glycoprotein, whereas a trans-dominant mutant did. Third, the use of truncation and deletion mutants showed that the NH 2 -terminal half of the SU was both necessary and sufficient for full trans-dominant inhibition. Finally, we also showed that HTLV-2 has a similar NH 2 -terminal determinant of envelope precursor assembly. To our knowledge, this study is the first to demonstrate the involvement of the SU portion of retroviral glycoproteins in ER assembly of the precursor. This feature was probably obscured by the fact that mature TM glycoproteins of retroviruses have always been found as stable oligomers , whereas mature SU glycoproteins have usually been detected as monomers . It should be noted, however, that oligomers of mature SU glycoproteins were observed in some studies . Whatever the contribution of SU to the oligomerization of the mature envelope glycoproteins may be, our study brings to light an SU requirement for the ER assembly of the HTLV-1 envelope precursor, and underscores that domains involved in the ER assembly of precursor glycoproteins should not be directly inferred from those defined for the mature oligomers. The difficulties in interpreting structural data for proteins that adopt different conformations in their lifetimes are further exemplified by the study of the leucine zipper-like motif present in the TM. Retroviral envelopes undergo at least two successive oligomerization events: the first is the assembly of the precursor glycoprotein in the ER, while the second is the formation of the fusion-competent oligomer triggered by receptor recognition at the cell surface. Although it was first thought that the zipper motif might fold into a coiled coil in the context of the precursor molecule, the leading hypothesis now is that it drives only the second oligomerization event . Using an in vivo experimental strategy, our study with the HTLV-1 envelope glycoprotein further corroborates the idea that the leucine zipper-like domain is not required for ER assembly, since glycoproteins with deletions encompassing the corresponding region were still capable of intracellular association with the wt precursor. Collectively, our data indicate that the NH 2 -terminal domain of the HTLV-1 envelope glycoprotein determines its ER assembly at a step that is not the last one in the acquisition of transport competence. The phenotype exhibited by most of our HTLV-1 envelope mutants (i.e., transport deficiency despite competence for oligomeric assembly) was previously observed with mutants of HA0 , VSV G , and the envelope of the Moloney murine leukemia virus . Thus, oligomerization per se is not sufficient to meet the quality control of the ER. The possibility of additional events in the maturation process was also suggested by the observation of a significant time lag between the ER dimerization of the HIV-1 envelope precursor and its Golgi cleavage . What is the exact nature of these postoligomerization events? It is possible that significant folding of the subunits proceeds within the framework of the oligomer. Consistent with this hypothesis, the HIV-1 envelope precursor has been found to acquire reactivity to stringent conformational antibodies only after its oligomeric assembly . Studies on the reovirus cell attachment protein σ1, a cytosolic protein, have revealed a schema of protein maturation that may also apply to membrane proteins in the ER. The maturation of this protein involves an initial trimerization of the NH 2 terminus that, in turn, permits the trimerization of the COOH terminus and completion of folding; the trans-dominant negative effects exerted by COOH-terminally truncated mutants led to the proposal that the folding of the NH 2 -terminally assembled subunits is a cooperative process requiring the integrity of all subunits . A similar mechanism is likely to account for the trans-dominant negative effects observed in our study of the HTLV-1 glycoprotein. It is indeed noteworthy that not only the heterodimers formed by the wt and any of the transport-defective mutants but also those formed by the wt and any of the COOH-terminally truncated, transport-competent mutants were defective in transport. Together with previous findings, our study thus emphasizes that the condition imposed on membrane proteins by the cellular quality control is not simply the acquisition of an oligomeric status as such, but rather the attainment of the correct quaternary structure. We propose that this process involves the cooperative folding of “preassembled” subunits. | Study | biomedical | en | 0.999996 |
10189369 | Yeast strains and plasmids are listed in Table I . Experiments with S . cerevisiae were carried out using strain DBY1034 and derivatives thereof. DBY1034 was transformed with plasmid pOH to obtain expression of Och1p-HA. Strain DBY1034-S13G, in which the endogenous SEC13 gene has been replaced with SEC13-GFP , was constructed as follows. The URA3 cassette from pUC1318-URA3 was excised with HindIII, blunted, and inserted into the SspI site of pUC19 to create pUC19-URA3. An 1,166-bp HincII-HindIII fragment spanning the 3′ portion of SEC13 was then amplified by PCR from S . cerevisiae genomic DNA and inserted into the corresponding sites in pUC19-URA3. The resulting plasmid was mutagenized using the QuikChange kit (Stratagene Inc.) to replace the SEC13 stop codon with a SnaBI site. The EGFP gene was excised from pEGFP-1 ( Clontech ) with BamHI and NotI, blunted, and inserted into this SnaBI site. The resulting construct was linearized at the unique BstEII site and integrated into the chromosomal SEC13 gene. This “pop-in” strain was then plated on 5-fluoroorotic acid to select for the “pop-out” recombinant strain DBY1034-S13G. Strain DBY1034-S23G, in which the endogenous SEC23 gene has been replaced with SEC23-GFP , was constructed in the same manner, beginning with the insertion into pUC19-URA3 of a 1,120-bp AflII-HindIII fragment spanning the 3′ portion of SEC23 . Similar strategies were used to replace the endogenous SEC24 and SEC31 genes with EGFP fusion genes. To construct strain DBY1034-S12m, in which the endogenous SEC12 gene has been replaced with SEC12-myc , a 1,685-bp HincII-XbaI fragment spanning the 3′ portion of SEC12 was inserted into pUC19-URA3; a c-myc epitope sequence was then inserted before the stop codon using the QuikChange kit, and the resulting construct was linearized with SspI for “pop-in” integration into the SEC12 locus. Experiments with P . pastoris were carried out using the prototrophic wild-type strain PPY1 or the isogenic his4 arg4 auxotroph PPY12 and derivatives thereof (Table I ). General methods for growth and transformation of P . pastoris have been described elsewhere . Strain PPY12-OH, which expresses Och1p-HA, was constructed as follows. A modified gene encoding tagged Och1p was excised from plasmid pOCHFT (a gift of Sean Munro, Medical Research Council, Cambridge, UK) by digesting at an upstream HindIII site (sequence including the start codon: AAGCTTAGAGATCATG), blunting, and digesting at an XbaI site immediately after the stop codon. This fragment was subcloned into pIB2 that had been digested with SmaI and SpeI. The resulting plasmid was digested with BstEII and PstI, and the corresponding BstEII-PstI fragment from pOH was inserted to create pIB2-OH, in which a gene encoding triple-hemagglutinin epitope (HA)–tagged Och1p is downstream of the strong constitutive GAP promoter. pIB2-OH was linearized with SalI and integrated into the his4 locus of PPY12. Strain PPY12-S13G, in which the endogenous SEC13 gene has been replaced with SEC13-GFP , was constructed as follows. pSG464 was digested with SnaBI and NdeI, blunted, and religated to yield pUC19-ARG4. This plasmid lacks the PARS2 sequence present in pSG464, and therefore can only transform P . pastoris by integration. A 461-bp HindIII-NsiI fragment spanning the 3′ end of SEC13 was then amplified by PCR from P . pastoris genomic DNA and inserted into pUC19-ARG4 that had been cut with HindIII and PstI. This plasmid was mutagenized to replace the SEC13 stop codon with a SnaBI site, and the EGFP gene was inserted into this site as described above. The resulting construct was linearized at the unique MscI site and integrated into the chromosomal SEC13 gene, yielding an intact SEC13-GFP fusion gene plus a 3′ fragment of authentic SEC13 . A similar strategy was used to create strain PPY12-S12m: a 1,328-bp BamHI fragment spanning the 3′ end of SEC12 was inserted into pUC19-ARG4; a c-myc epitope sequence was then inserted just upstream of the HDEL sequence, and the resulting construct was linearized with XhoI for integration into the SEC12 locus. To express myc-tagged S . cerevisiae Mnt1p in P . pastoris , the gene encoding tagged Mnt1p was excised from pYMT1BS (a gift of Sean Munro) and subcloned into pOW3, a P . pastoris episomal vector that contains the GAP promoter . Plasmid pAFB584, which encodes glutathione S -transferase fused to the NH 2 -terminal acidic domain of S . cerevisiae Sec7p (up to the NaeI site in the coding sequence), was provided by Alex Franzusoff (University of Colorado, Denver, CO). P . pastoris cultures were grown overnight at 30°C to an OD 600 of ∼0.15 in 1% yeast extract, 2% peptone, 2% glucose, 20 mg/liter adenine sulfate, 20 mg/liter uracil, 50 mM sodium maleate, pH 5.5. Half of a culture then received nocodazole at a final concentration of 15 μg/ml, diluted from a 10 mg/ml stock solution in water-free dimethyl sulfoxide. As a control, the other half of the culture received only dimethyl sulfoxide. Incubation was continued for up to 2.5 h, and the cells were fixed for microscopy. Anti–HA monoclonal antibody (16B12; Berkeley Antibody Co.), anti– myc monoclonal antibody (9E10; Boehringer Mannheim Biochemicals ) and anti–green fluorescent protein (GFP) monoclonal antibody (a mixture from clones 7.1 and 13.1; Boehringer Mannheim Biochemicals ) were used at 5 μg/ml; in cells not expressing a tagged protein, each antibody gave only a faint background signal. The anti–GFP antibody was used to supplement the endogenous GFP signal, which is diminished by the immunofluorescence procedure. Anti–β-tubulin monoclonal antibody (KMX-1; Boehringer Mannheim Biochemicals ) was used at 1 μg/ml. The following rabbit polyclonal antibodies were used. Anti–Pdi1p serum (a gift of Peter Walter, University of California, San Francisco, CA; originally produced by Victoria Hines, Chiron Corp., Emeryville, CA), raised against SDS-PAGE–purified S . cerevisiae Pdi1p, was used at a dilution of 1:350. Alex Franzusoff generously provided an antiserum raised against a fusion between β-galactosidase and the NH 2 -terminal portion of Sec7p ; this antibody was used at a dilution of 1:500. Oregon green 488–conjugated goat anti–mouse IgG and Texas red-X–conjugated goat anti–rabbit IgG (Molecular Probes, Inc.) were used at 20 μg/ml. Controls were performed (not shown) to check the specificity of the polyclonal antibodies. In the case of anti–Pdi1p, the immunofluorescence signal could be quenched by preincubating with a protein fragment (a gift of Robert Freedman, University of Kent at Canterbury, UK) comprising the COOH-terminal 308 residues of S . cerevisiae Pdi1p. The anti–Sec7p antibody specifically recognized a glutathione S -transferase–Sec7p fusion protein on an immunoblot of an extract from Escherichia coli cells expressing this protein. Many rabbit antisera contain traces of reactivity against α-1,6-mannose linkages, and because S . cerevisiae and P . pastoris both add α-1,6-mannose residues to glycoproteins transiting through the Golgi , these contaminating antibodies give a spurious labeling of Golgi structures (not shown). With S . cerevisiae , anti–α-1,6-mannose antibodies label Golgi cisternae in immunoelectron microscopy experiments ; and when used at very high dilutions for immunofluorescence, such antibodies primarily label early Golgi elements. Similarly, with P . pastoris , anti–α-1,6-mannose antibodies give a strong and relatively specific Golgi labeling. In several cases, we observed punctate staining with antisera raised against various COPII proteins, but this staining was actually due to contaminating anti–α-1,6-mannose antibodies. This problem can be avoided by preincubating a diluted antibody either with fixed S . cerevisiae cells or with 0.5 mg/ml purified yeast mannan . Thin-section electron microscopy was performed essentially as described . In brief, a 50-ml culture of yeast cells in rich glucose medium was grown to an OD 600 of ∼0.5. The culture was concentrated to a volume of <5 ml with a bottle-top vacuum filter, and 40 ml of ice-cold 50 mM KP i , pH 6.8, 1 mM MgCl 2 , 2% glutaraldehyde was added rapidly with swirling. After fixation for 1 h on ice, the cells were washed repeatedly, and then resuspended in 0.75 ml 4% KMnO 4 and mixed for 1 h at room temperature. The cells were washed, and then resuspended in 0.75 ml 2% uranyl acetate and mixed for 1 h at room temperature. Finally, the cells were embedded in Spurrs resin; 50 ml of yeast culture yielded enough cells for three BEEM capsules. The resin was polymerized for 2 d at 68°C. Sections were stained with uranyl acetate and lead citrate, and viewed on an electron microscope (100 CXII; JEOL U.S.A. Inc.). For immunoelectron microscopy, we used a modification of previously published methods . A 50-ml log-phase culture was concentrated to a volume of <5 ml (see above) and fixed by adding 40 ml of 50 mM KP i , pH 6.8, 1 mM MgCl 2 , 4% formaldehyde, 0.1% glutaraldehyde (aldehydes were EM grade from Ted Pella, Redding, CA). After 1 h at room temperature, the cells were washed twice with 20 ml PBS, pH 7.4, containing 0.5% 2-mercaptoethanol, 0.5 mM o -phenanthroline, 0.25 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, and then once more with 1 ml of the same solution. Treatment with periodate was omitted because this compound may destroy certain antigens by reacting with amino acid side chains . The washed cells were embedded in 100 μl of low-melting–temperature agarose, which was cut into cubic-millimeter blocks and infiltrated for 2 h at room temperature in 1.7 M sucrose, 25% polyvinylpyrrolidone K15 (Fluka Chemical Corp.) in PBS, prepared according to Tokuyasu . Individual agarose blocks were then mounted on copper pins, frozen in liquid nitrogen, and cryosectioned at −80°C. Thawed cryosections were prepared and labeled as described , using a blocking buffer consisting of PBS, 20 mM glycine, 1% fish gelatin ( Sigma Chemical Co. ), and 10% fetal calf serum; the serum had been heated at 60°C for 1 h, and then centrifuged to remove complement. Dilutions were 1:10 for an affinity-purified polyclonal anti–HA antibody (a gift of Jan Burkhardt, University of Chicago, Chicago, IL), 1:25 for an affinity-purified polyclonal anti–GFP antibody (a gift of Charles Zuker, University of California, San Diego, San Diego, CA), and 1:50 for protein A-gold (10 nm; Goldmark Biologicals). After the protein A-gold step, the sections were postfixed for 30 min in 1% glutaraldehyde in PBS, and then washed with distilled water and stained with 1.5% silicotungstic acid in 2.5% polyvinyl alcohol (MW 15,000; ICN Biomedicals Inc.). The best results were obtained using relatively thick sections and a thin layer of stain. Electron micrographs were digitized and imported into Photoshop (Adobe Systems Inc.) to adjust brightness and contrast and to create composite images. For Figs. 3 and 7 , the gold particles were darkened to improve visualization. Images were printed on an NP-1600 dye-sublimation printer (Codonics). We modified previously described methods to enhance both the preservation and the visualization of intracellular structures. Improved preservation was achieved by rapidly digesting the cell wall with protease-free lyticase in sorbitol-free wash buffer, and by postfixing the cells in acetone. Improved visualization was achieved by adhering the cells directly to coverslips rather than to wells on a slide. A detailed protocol follows. A yeast culture is grown overnight in rich or selective medium with good aeration to an OD 600 of 0.25–1.0. 8 ml of this culture is pipetted onto a 150-ml bottle-top filter, and the liquid is removed by vacuum. The cells are immediately resuspended in 5 ml of freshly prepared 50 mM KP i , pH 6.5, 1 mM MgCl 2 , 4% formaldehyde (EM grade; Ted Pella). After fixation for 2 h at room temperature in a 15-ml tube (Falcon Labware), the cells were centrifuged for 3 min at 1,000 g (2,000 rpm) in a tabletop centrifuge, and the supernatant is aspirated completely with a Pasteur pipet. The cells are resuspended in 5 ml of freshly prepared wash buffer (100 mM KP i , pH 7.5, 1 mM MgCl 2 , 0.25 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin), and then centrifuged again as above. Finally, the cells are resuspended in wash buffer to an OD 600 of 10. To 100 μl of concentrated cell suspension is added 0.6 μl of 2-mercaptoethanol followed by 20 μl of recombinant yeast lytic enzyme (20,000 U/ml; ICN Biomedicals Inc.). After mixing end-over-end for 15–30 min at room temperature, the spheroplasts are centrifuged for 2 min at 400 g (2,000 rpm) in a microfuge, gently resuspended in 100 μl wash buffer, and then centrifuged and resuspended once again. To create “wells” on a coverslip, a suitable piece is cut from the adhesive backing of an express courier document pouch, holes are made with a rotating-head hole punch, and the perforated plastic is attached to the coverslip. 10 μl of 0.1% polylysine is added to each well, and then removed by vacuum aspiration. Each well is washed three times with water, and 10 μl of spheroplast suspension is added. After 3 min, excess liquid is blotted off with a cotton swab, and the dried coverslip is immersed in 40 ml of acetone precooled to −20°C in a 50-ml tube (Falcon Labware). (This organic solvent fixation step is important for preserving tER and Golgi structure. For some antigens methanol works better, but in most cases acetone is superior.) After 5 min, the coverslip is removed, inverted onto a paper towel to blot off excess solvent, and allowed to dry. Each well is precoated for 30 min with a drop of PBS-Block (PBS, pH 7.4, 1% dried milk, 0.1% bovine serum albumin, 0.1% octyl glucoside). 10 μl of primary antibody mixture in PBS block is then added, and the coverslip is incubated in a humid chamber for 1 h. Each well is washed eight times with PBS block, and 10 μl of secondary antibody mixture in PBS block is added. 2 μg/ml 33258 (Hoechst AG) is included in the secondary antibody solution to stain DNA. After a 30-min incubation in the dark, each well is washed eight times with PBS block. The liquid is aspirated completely after the final wash. Each well then receives 5 μl of phenylenediamine-containing mounting medium . The coverslip is inverted onto a slide and sealed with nail polish. Samples were viewed on an Axioplan microscope equipped with a 100× Plan-Apo 1.4 NA objective ( Carl Zeiss, Inc. ) and with band-pass filters for visualizing Hoechst dye, fluorescein/Oregon green, and Texas red. Images were captured with a Photometrics CCD camera using the Openlab software package (Improvision). For each field of cells, multiple images were recorded from focal planes spaced 0.2 μM apart; three to four of these images were deblurred using a simple deconvolution algorithm, and then combined to form a single projected image. Where indicated, the same cells were also photographed in differential interference contrast (DIC) mode at a single focal plane. Photoshop was used for colorization, adjustments of brightness and contrast, and creation of composite and merged images. Manipulations with the Openlab and Photoshop programs improved the quality of the images, but did not significantly alter their information content. Fluorescence and DIC images were printed on a Stylus Photo color inkjet printer (Epson Electronics America Inc.). To quantify the overlap between Och1p-HA and Sec7p, digital images were examined with Photoshop. Cells that showed clear staining with both antibodies were used for quantitation. A spot of one color was scored as overlapping with a spot of the second color if the center of the first spot was contained within the second spot. To estimate the percentage of Golgi structures that are perinuclear in P . pastoris , the perinuclear and peripheral Och1p-HA spots were counted in 50 cells that showed faint nuclear envelope staining . To estimate the average percentage of the cell area occupied by fluorescent spots of a given marker protein, 25 cells stained to reveal Och1p-HA and Sec7p were analyzed using NIH Image ( http://rsb.info.nih.gov/nih-image/ ): the total areas of the Och1p-HA and Sec7p spots were summed and divided by twice the total areas of the cells. Yeast cells expressing a GFP-labeled COPII protein were grown to log phase with good aeration. A 400-μl aliquot of the culture was then mixed rapidly with 400 μl of 100 mM KP i , pH 6.5, 2 mM MgCl 2 , 8% formaldehyde, 0.5% glutaraldehyde. After fixation for 1 h in the dark, the cells were washed twice with 1 ml PBS, and then resuspended in 50 μl PBS. For viewing by fluorescence microscopy, 1–2 μl of the resuspended cells were spotted on a slide and spread with a coverslip. Stacks of Golgi cisternae can be readily visualized in P . pastoris cells, with each stack containing about four cisternae . Previously published images of P . pastoris showed Golgi stacks near the nucleus . Golgi structures might form by growing out of the nuclear envelope, which constitutes a large fraction of the ER in budding yeasts . Alternatively, P . pastoris Golgi stacks might be positioned near the nucleus by microtubule-dependent transport toward the centrosome, as occurs in vertebrate cells . Such a role for microtubules is excluded by the following two observations. First, thin-section electron microscopy indicates that a typical P . pastoris cell contains several distinct Golgi stacks, only some of which are located near the nucleus . Other Golgi stacks are found next to the peripheral ER elements that underlie the plasma membrane. Second, treatment of P . pastoris cells with nocodazole does not visibly alter the structure or positioning of Golgi stacks . As with S . cerevisiae , nocodazole treatment of P . pastoris disrupts microtubules and inhibits nuclear division , but Golgi stacks are still observed next to the nuclear envelope and peripheral ER. In both untreated and nocodazole-treated cells, vesicular profiles are frequently seen in ER regions adjacent to the Golgi cisternae . These vesiculating ER regions resemble the tER sites seen in vertebrate cells . Hence, the morphological data suggest that Golgi stacks in P . pastoris are associated not with the centrosome, but rather with tER sites. Electron microscopy indicates that general ER structure is similar in P . pastoris and S . cerevisiae , but that Golgi organelles in P . pastoris are more coherent (see above). We confirmed this interpretation by visualizing various marker proteins (Table I ) using immunofluorescence microscopy. For these experiments, we developed a modified immunofluorescence protocol that consistently yields high-quality images (see Materials and Methods). Because P . pastoris is closely related to S . cerevisiae , polyclonal antibodies raised against S . cerevisiae antigens often cross-react with the P . pastoris homologues. Alternatively, marker proteins were modified with epitope tags and visualized using specific monoclonal antibodies. To visualize the general ER in P . pastoris , fixed cells were labeled with an antibody against protein disulfide isomerase (Pdi1p), a marker for the general ER . The same cells were also incubated with Hoechst dye to label DNA. The anti–Pdi1p antibody highlights the nuclear envelope as well as peripheral ER elements. This pattern resembles the ER distribution seen in S . cerevisiae . Fig. 2 A shows the S . cerevisiae Golgi. This organelle appears in immunofluorescence images as a set of punctate spots, with different Golgi markers often displaying only a partially overlapping localization . In S . cerevisiae cells expressing an HA-tagged version of the early Golgi marker Och1p , an anti–HA monoclonal antibody reveals multiple spots of Och1p-HA labeling per cell. The same cells were also labeled with a polyclonal antibody (red) against Sec7p, a protein that is concentrated in late Golgi elements . The two markers show no significant overlap . Do early and late Golgi elements ever colocalize in S . cerevisiae ? In temperature-sensitive sec14 mutants of S . cerevisiae , multilamellar Golgi structures accumulate at the nonpermissive temperature ; these structures are reminiscent of the Golgi stacks seen in higher eukaryotes. Therefore, we tested whether Och1p-HA and Sec7p colocalize in a sec14 mutant after incubation at the nonpermissive temperature of 37°C. The 37°C treatment causes redistribution of Sec7p into large clusters . However, the staining pattern of Och1p-HA is largely unchanged in the sec14 mutant , and there is still no significant overlap between Och1p-HA and Sec7p. It seems that S . cerevisiae is incapable of generating coherent Golgi stacks that contain both early and late Golgi proteins. Fig. 2 C shows the P . pastoris Golgi. Using an integrating expression vector , we generated a P . pastoris strain that stably expresses HA-tagged S . cerevisiae Och1p. P . pastoris contains an α-1,6-mannosyltransferase activity like that ascribed to Och1p , and expression of Och1p-HA in P . pastoris has no effect on growth or Golgi morphology (not shown). Och1p-HA localizes to two to six spots per P . pastoris cell . As predicted from electron microscopy, a subset (∼45%) of the Och1p-HA spots clearly adjoin the nucleus . When P . pastoris cells are labeled with the antibody against S . cerevisiae Sec7p, two to six spots are again detected , presumably because this antibody reacts with the P . pastoris homologue of Sec7p. By contrast to the situation in S . cerevisiae , Och1p-HA and Sec7p exhibit nearly quantitative overlap in P . pastoris . Similarly, when a tagged version of the S . cerevisiae medial-Golgi protein Kre2p/Mnt1p is expressed in P . pastoris , it colocalizes with Sec7p (not shown). Colocalization at the light microscopy level indicates that two markers are either in the same compartment or in closely apposed structures. These data support the conclusion that Golgi organelles are more coherent in P . pastoris than in S . cerevisiae . To confirm that the structures visualized in P . pastoris by immunofluorescence are indeed Golgi membranes, we performed immunoelectron microscopy on thawed cryosections. Using a polyclonal anti–HA antibody to detect Och1p-HA, we see a specific labeling of Golgi stacks . P . pastoris resembles vertebrate cells in having stacked Golgi cisternae. We tested whether the two cell types are also similar with regard to Golgi dynamics. Pre-Golgi elements in vertebrate cells are transported along microtubules , but our electron microscopy data indicate that in P . pastoris , microtubules do not influence Golgi structure . This conclusion was confirmed at the immunofluorescence level. In untreated P . pastoris cells, microtubules are present and nuclei partition into daughter cells during mitosis . Microtubule distribution shows no detectable relationship to Golgi distribution . Nocodazole treatment depolymerizes microtubules and blocks nuclear migration, but the Golgi staining pattern is unaffected . Moreover, the Golgi markers Sec7p and Och1p-HA still colocalize in nocodazole-treated P . pastoris cells (not shown). To determine whether cell cycle progression alters Golgi organization in P . pastoris , as it does in vertebrate cells , we analyzed P . pastoris in the G1, S/G2, and M phases of the cell cycle. Immunofluorescence was used to quantify the average number of Golgi structures per cell. This number is similar in each phase of the cell cycle (Table IV ). Thus, when observing P . pastoris under a variety of conditions, we consistently find that each cell contains a small number of distinct Golgi stacks. The electron microscopy data suggest that P . pastoris contains discrete tER sites, whereas S . cerevisiae lacks such sites. To explore this idea further, we compared the localizations of COPII proteins in the two yeasts. GFP was fused to the COOH terminus of S . cerevisiae Sec13p, a coat protein that is incorporated at a late stage of COPII vesicle assembly . Replacement of the endogenous SEC13 gene with the SEC13-GFP gene has no detectable effect on cell growth, indicating that the Sec13p-GFP fusion can perform the essential function of Sec13p . When S . cerevisiae cells expressing Sec13p-GFP are viewed by fluorescence microscopy, each cell contains ∼30–50 tiny spots . The spots are distributed almost evenly throughout the cytoplasm, with some cells showing an apparent concentration of spots on the nuclear envelope. We surmise that these spots represent individual COPII vesicles. A vesicle is smaller than the resolution limit of light microscopy , so the apparent size of the spots is probably misleading, but a single COPII vesicle contains many copies of Sec13p-GFP and hence should produce a detectable fluorescence signal. GFP was also fused to three other S . cerevisiae COPII coat proteins : Sec23p, Sec24p, and Sec31p. All of these fusions are functional, and they all give the same fluorescence pattern as Sec13p-GFP , indicating that we have visualized the normal distribution of COPII vesicles. These results strongly support the notion that COPII vesicles bud from the entire ER in S . cerevisiae . In parallel, the P . pastoris SEC13 gene was replaced with a SEC13-GFP fusion gene. The resulting fluorescence pattern is strikingly different from that seen in S . cerevisiae . Sec13p-GFP in P . pastoris is concentrated in only two to six large spots per cell . When examined by immunofluorescence microscopy, the Sec13p-GFP spots are adjacent to, but not quite overlapping, the Golgi spots marked by the anti–Sec7p antibody . This result suggests that Sec13p-GFP is localized to tER sites. Indeed, immunoelectron microscopy with an anti–GFP antibody revealed that Sec13p-GFP is present on tubulovesicular structures at the interface between ER membranes and Golgi stacks . We conclude that COPII vesicle budding is restricted to discrete tER sites in P . pastoris . The earliest known player in the COPII assembly pathway is Sec12p, a membrane-bound guanine nucleotide exchange factor that recruits the small GTPase Sar1p to the ER membrane . In previous studies of S . cerevisiae , Sec12p exhibited general ER staining . Our results confirm those earlier findings. S . cerevisiae Sec12p has traditionally been visualized in strains overexpressing this protein . To eliminate possible ambiguities resulting from overexpression, we replaced the chromosomal SEC12 gene with a myc-tagged version. The resulting strain grows like the wild-type (not shown), implying that Sec12p-myc can functionally replace the essential wild-type protein . Sec12p-myc colocalizes with Pdi1p in the nuclear envelope and in peripheral ER membranes. Although Sec12p-myc sometimes shows a discontinuous staining pattern, the fluorescence signal is relatively weak, and we have observed that the ER network often appears discontinuous when it is weakly stained (not shown). Hence, the combined data suggest that Sec12p-myc is present throughout the ER in S . cerevisiae . We also replaced P . pastoris SEC12 with a myc-tagged version. Once again the fluorescence signal is weak, but the pattern is clearly visible. In this case, the anti–myc antibody does not give a general ER staining, but instead labels several spots per cell . Like Sec13p-GFP (see above), Sec12p-myc localizes to sites that are immediately adjacent to Sec7p-containing Golgi structures . Thus, in P . pastoris , components at both early and late stages of the COPII assembly pathway are concentrated at tER sites. Why is the Golgi apparatus more photogenic in P . pastoris than in S . cerevisiae ? These two yeasts are morphologically very similar; yet Golgi cisternae in P . pastoris are organized into stacks, whereas Golgi cisternae in S . cerevisiae are scattered throughout the cytoplasm. We propose the following hypothesis. In P . pastoris , COPII vesicles bud from fixed tER sites, and then fuse with one another to create new Golgi cisternae, which mature to yield polarized stacks . In S . cerevisiae , COPII vesicles bud throughout the ER, and therefore each Golgi cisterna forms at a different location . Our immunofluorescence data confirm that S . cerevisiae and P . pastoris have fundamentally different Golgi structures. A hallmark of the Golgi in S . cerevisiae is that various marker proteins often show distinct punctate distributions . For example, the early Golgi protein Och1p-HA exhibits virtually no overlap with the late Golgi protein Sec7p. Although multilamellar Golgi structures are seen in temperature-sensitive sec7 and sec14 mutants of S . cerevisiae , we find that early and late Golgi markers still do not colocalize in sec14 mutant cells , indicating that S . cerevisiae cannot make coherent Golgi stacks. With P . pastoris , on the other hand, Och1p-HA and Sec7p overlap almost completely , as expected if each Golgi stack represents an ordered set of early, middle, and late cisternae. Does this difference in Golgi structure correlate with a difference in ER organization? The ER of both yeasts comprises the nuclear envelope plus peripheral elements. However, in P . pastoris , vesicles can often be seen budding specifically from regions of the ER adjacent to Golgi stacks . Such vesiculating ER regions have not been seen in S . cerevisiae . These observations suggested to us that P . pastoris contains discrete tER sites, whereas S . cerevisiae does not. To test this interpretation, we used COPII coat proteins as markers for the tER . With S . cerevisiae , fusing GFP to Sec13p or other COPII coat proteins revealed many small fluorescent spots that probably represent individual COPII vesicles . These spots are found throughout the cytoplasm, consistent with the notion that COPII vesicles bud at random from the entire ER. By contrast, a Sec13p-GFP fusion in P . pastoris localizes to a small number of discrete regions . These regions are immediately adjacent to Golgi stacks, and they contain tubulovesicular membranes . Hence, we propose that Sec13p-GFP marks a compartment in P . pastoris that is analogous to the tER sites described previously in vertebrate cells . How is Sec13p recruited to tER sites in P . pastoris ? The assembly of COPII vesicles is an ordered process that begins with the action of Sec12p . We compared the localization of epitope-tagged Sec12p in the two yeasts. Consistent with previous reports , Sec12p-myc in S . cerevisiae is distributed throughout the ER. In P . pastoris , on the other hand, Sec12p-myc is concentrated at tER sites . This observation suggests a working model for the organization of tER and Golgi compartments in the two yeasts. We postulate that in P . pastoris , Sec12p is anchored at tER sites by unknown partner proteins that comprise a “tER scaffold.” Because Sec12p initiates the assembly of COPII vesicles, these vesicles bud exclusively from tER sites, and successive Golgi cisternae form at fixed locations to generate polarized stacks. This model assumes that tER sites are relatively stable entities, and, indeed, our studies of Sec13p-GFP dynamics in P . pastoris confirm that tER sites are long lived and slow moving (B.J. Bevis, unpublished observations). The situation is quite different in S . cerevisiae— this yeast apparently lacks a tER scaffold, so Sec12p is free to diffuse throughout the ER. As a result, COPII vesicles bud from the entire ER, and successive Golgi cisternae form at different locations, yielding a dispersed organelle . In this view, Golgi structure and positioning are strongly influenced by tER organization. An important test of our model will be to alter tER organization in P . pastoris and ask whether Golgi structure is correspondingly affected. Such an experiment will reveal whether the existence of Golgi stacks in P . pastoris is due solely to the presence of fixed tER sites. The simplest view is that Golgi stacking is a kinetic phenomenon, with cisternal formation and maturation occurring too quickly for successive cisternae to diffuse away from one another. Alternatively, Golgi cisternae in P . pastoris might be held together by a cytosolic matrix . These possibilities can be distinguished by generating P . pastoris mutants in which COPII proteins are delocalized, and then asking whether the loss of tER sites leads to Golgi dispersal. One promising approach focuses on determining whether P . pastoris Sec12p contains a tER localization signal that is recognized by specific partner proteins. Although S . cerevisiae lacks discrete tER sites, this yeast can be used to explore the more general question of whether the Golgi is an outgrowth of the ER. Early in S-phase of the S . cerevisiae cell cycle, the small buds invariably contain both ER and Golgi structures . It is likely that the ER elements present in the emerging bud give rise to new Golgi cisternae. If so, mutants that fail to transport ER membranes into the bud should also lack Golgi cisternae in the bud. We are currently testing this prediction by characterizing S . cerevisiae mutants defective in Golgi inheritance. A comparison of budding yeasts with other eukaryotes can indicate which aspects of the tER-Golgi system are cell type–specific. First, unlike many eukaryotes, S . cerevisiae contains neither stacked Golgi organelles nor discrete tER sites. It is unclear whether the absence of these structures in S . cerevisiae is adaptive, or whether it reflects a loss-of-function mutation during the evolution of this yeast. Second, tubular connections between Golgi stacks are present in vertebrate cells , but have not been detected in P . pastoris , the fission yeast Schizosaccharomyces pombe or certain insect cells . Third, the Golgi breaks down during mitosis in vertebrate cells , but not in higher plants or budding yeasts (Table IV ) . Which aspects of the tER-Golgi system are universal? We propose that Golgi cisternae always form by the coalescence of tER-derived membranes. This relationship is particularly evident in cells that contain Golgi stacks immediately adjacent to tER sites . We have now documented such an association in P . pastoris . A close apposition between tER sites and Golgi stacks is probably a general feature of cells that do not transport Golgi elements along cytoskeletal tracks. Consistent with this idea, microtubules have no influence on Golgi structure or positioning in P . pastoris . However, in some cell types, nascent Golgi elements are transported away from tER sites, thereby obscuring the tER-Golgi connection. For example, in S . pombe , microtubules play a role in cisternal stacking and possibly in Golgi movement . In higher plants, Golgi structures are transported along actin filaments . The best-studied example of cytoskeleton-mediated Golgi movement is provided by vertebrate cells, which employ microtubules and associated motor proteins to generate a juxtanuclear Golgi ribbon . As a consequence, tER sites and Golgi elements normally do not colocalize in vertebrate cells. Yet recent evidence suggests that nascent Golgi structures in vertebrate cells initially coalesce at tER sites ; in the presence of microtubule-depolymerizing agents, entire Golgi stacks are found next to tER sites . Thus, when microtubules have been disrupted, the tER-Golgi system in a vertebrate cell resembles the tER-Golgi system in P . pastoris . It seems that in all eukaryotes, the Golgi can be viewed as a dynamic outgrowth of the tER. | Study | biomedical | en | 0.999997 |
10189370 | HeLa and COS-7 cells were grown under standard conditions. IgG fraction (10 mg/ml final concentration) from RM autoimmune serum (AS) and affinity-purified anti–GMAP-210 antibodies were obtained as described in Rios et al. . CTR433, a mouse monoclonal antibody, is a marker of medial-Golgi . Polyclonal antibody anti– γ-tubulin has been previously characterized . Anti-detyrosinated tubulin (αT12 and SG) and anti-p115 antibodies were supplied by Drs. Kreis, Bulinski, and Sztul, respectively. 10 6 recombinants of a λZAPII human HeLa cell random-primed cDNA expression library (P. Chambon, IGBMC, Universitè Louis Pasteur, Strasboug, France) were screened with the autoimmune serum diluted 1:1,500 followed by anti–human IgG alkaline phosphatase conjugated ( Promega Corp. ), and positive plaques were detected by incubation with BCIP and NBT. Positive clones were plaque purified through several rounds of rescreening. Affinity-purified antibodies against each of the three major autoantigens recognized by the serum were obtained and used to group the clones obtained in immunologically related families. pBluescript phagemid containing the cloned cDNA inserts were excised from λZAPII by coinfection with R408 helper phage as described in the manufacturer's instructions (Stratagene). The 3′ end of GMAP-210 was cloned by RT-PCR. HeLa mRNA was prepared with a Quick prep mRNA purification kit ( Pharmacia Biotech ). First-strand cDNA synthesis was performed with an oligo-(dT) primer by using a First-strand cDNA synthesis kit ( Pharmacia Biotech ). The completed first-strand reaction was amplified directly by PCR with specific primers. Clone RT-10, containing the 3′ end of GMAP-210 cDNA, was obtained with the primers P1 and P2 as indicated in Fig. 1 A. A series of overlapping restriction fragments from the clones obtained by immunoscreening were subcloned into pBluescript SK or pTZ19R. Double-stranded cDNA in pBluescript or pTZ19R was sequenced on both strands with an automatic sequencer ( Pharmacia ) by the dideoxy termination method . Sequence data were compiled and analyzed using the University of Wisconsin Genetics Computer Group package version 8.1 for Unix computers and the EGCG extensions to the Wisconsin package version 8.1.0. Comparisons to known sequences were performed using tFASTA and BLAST. Primary and secondary structure analysis was conducted with Motifs, PeptideStructure, PepStats, and PepCoil. The full-length open reading frame of GMAP-210 was assembled in the eukaryotic expression vector pECE using clones R2, R3, RT-7 (obtained by RT-PCR using primers P3 and P4), R6, and RT-10 . To obtain an HA epitope-tagged GMAP-210 protein, primers P5 and P6 were used to create an EcoRI site in the appropriate frame for cloning into pECE-HA. GMAP-210 cDNA fragments coding for amino acids 1–375 (ΔC375) and 1,778–1,979 were cloned in fusion with the green fluorescent protein (GFP) into the eukaryotic expression vectors pEGFPN1 and pEGFPC1, respectively. Full-length or partial GMAP-210 cDNAs were also cloned into the prokaryotic expression plasmids pGEX. The synthesis of the glutathione- S -transferase (GST) fusion proteins was induced by addition of 1 mM isopropyl-β- d -thiogalactoside and the fusion proteins were isolated from bacterial lysates by affinity chromatography with glutathione-agarose beads ( Sigma ). Northern analysis was performed on poly(A) + RNA from human tissues (commercial multiple Northern blot; Clontech ). DNA fragments corresponding to nucleotides 418–1,130 and 5,364-5,800 were radiolabeled and used as probes. After cloning DNA fragments coding for amino acids 375–611 or 618–803 into pGEX vectors, GST-fusion polypeptides were expressed and purified as described above. Fusion proteins were dialyzed in PBS and two rabbit polyclonal antibodies were generated (Agrobio). Sera were collected and Ig fraction purified by ammonium sulfate precipitation. These anti– GMAP-210 polyclonal antibodies were named RM127 and RM130. Gel electrophoresis and immunoblotting (IB) were performed as described . For immunoprecipitation experiments, HeLa cells were incubated for 30 min on ice in order to depolymerize microtubules and then treated with 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 containing protease inhibitors. Soluble fraction was clarified at 15,000 g for 15 min and preadsorbed with 5 μl of normal human serum or 5 μl of each preimmune rabbit sera on 100 μl of protein A–Sepharose. GMAP-210 was immunoprecipitated from supernatants with 5 μl AS, 5 μl RM127, or 5 μl RM130 on 100 μl of protein A–Sepharose. After incubation and washing, the presence of GMAP-210 in the bead pellets was detected by IB with RM130 polyclonal antibody. 2 × 10 8 HeLa cells were trypsinized and harvested at 500 g for 10 min at room temperature. Subcellular fractionation in order to obtain a high speed supernatant (HSS) was carried out as described by Rickard and Kreis . The protein concentration of the HSSs obtained ranged between 6–8 mg/ml when measured using the Bradford assay. The HSS was adjusted at 3–4 mg/ml with PEM buffer (0.1 M K-Pipes, 2 mM EGTA, 1 mM MgSO 4 , pH 6.8), incubated for 30 min on ice and then incubated for 15 min at 37°C in the presence of 10 μM Taxol™ (Paclitaxel), 1 mM DTT, and 1 mM GTP. Microtubules were centrifuged at 30,000 g for 30 min at 30°C through a cushion of 10% sucrose in PEM containing 10 μM taxol, 1 mM DTT, and 1 mM GTP. Microtubules were washed and resuspended in the same buffer (0.1–0.05 times the original HSS volume). Proteins were eluted from the microtubules by adding 10 mM ATP, 10 mM GTP, 0.5 M NaCl, or 2 M urea, respectively, and incubation at 37°C for 30 min followed by centrifugation. Tubulin was purified from bovine brain by two cycles of polymerization followed by chromatography on phosphocellulose. 10–20 μg of pure tubulin/assay were polymerized with 20 μM taxol, 1 mM GTP, and 1 mM DTT for 15 min at 37°C. Microtubules were centrifuged through 10% sucrose, washed, and finally resuspended in PEM containing GTP and taxol. After cloning different DNA fragments into pGEX vectors, GST-fusion polypeptides were expressed and isolated from bacterial lysates by affinity chromatography with glutathione-agarose beads. Microtubules were then added to glutathione-agarose beads linked to similar amounts of GST alone, GST-fusion polypeptides or GST-GMAP-210 (<0.5 μg). After incubation for 2 h at room temperature, beads were low speed sedimented (1,000 g ) and washed in PEM buffer. The presence of microtubules in the bead pellets was detected with anti–α-tubulin antibodies. Alternatively, GMAP-210 was immunoprecipitated from HeLa cells NP-40 supernatants using 5 μl AS, 5 μl RM127, or 5 μl RM130 coupled to 100 μl of protein A–Sepharose as described in a previous section. After washing, beads were incubated for 2 h at room temperature with taxol-polymerized microtubules. Then, beads were low speed sedimented (1,000 g ) and washed in PEM buffer. The presence of microtubules in the bead pellets was detected with anti–α-tubulin antibodies. HeLa cells were incubated with 10 μM NZ or 10 μM taxol for 4 h at 37°C. Untreated, NZ- or taxol-treated HeLa cells were washed twice with PBS at room temperature and once with a microtubule-stabilizing buffer PM2G (0.1 M K-Pipes, 2 M glycerol, 5 mM MgCl 2 , and 2 mM EGTA, pH 6.9). Then, washed cells were extracted for 5 min at room temperature with 0.1–0.3% NP-40 in the same buffer to obtain NP-40 soluble fractions (fraction S). The detergent-extracted preparations, that remained attached to substrate, were washed with PM2G without detergent and incubated for 30 min at 4°C in the same buffer containing 5 mM CaCl 2 to depolymerize microtubules (fraction Ca). The detergent and calcium-insoluble preparations were also harvested (fraction I) and all fractions obtained analyzed by SDS-PAGE and IB. All buffers contained protease inhibitors (1 mM PMSF and 1 μg/ml each of aprotinin, pepstatin, chymostatin, and leupeptin). In the case of cells pretreated with drugs, each of the washes contained the same concentration of the drug. In parallel, HeLa cells grown on coverslips were treated and successively extracted with NP-40 and calcium as described above. Cell ghosts were then processed for immunofluorescence. The presence of microtubules was revealed by using an anti–α-tubulin antibody. Fractions enriched in Golgi membranes were isolated from HeLa cells by flotation in a sucrose gradient as described . Before further processing, Golgi-enriched fractions were diluted by addition of several volumes of 10 mM Tris-HCl, pH 7.4 and protease inhibitors were added. The suspensions were then centrifuged at 200,000 g for 2 h at 4°C. To obtain GMAP-210–depleted Golgi membranes, membrane pellets were incubated with 2 M NaCl for 30 min on ice, high speed centrifuged, and finally resuspended in 10 mM Tris-HCl, pH 7.4, containing 250 mM sucrose. GST-fusion polypeptides were purified on glutathione-agarose beads. Preliminary experiments suggested that the presence of GST in the NH 2 terminus of the fusion proteins could decrease the association of the recombinant proteins to membranes. Therefore, polypeptides were released from the fusion proteins by treatment with 1% thrombin (wt/wt). Full-length GMAP-210 and ΔN1597 construct could not be tested because they were degraded by thrombin treatment. Binding to Golgi membranes was performed using 10 μg of Golgi membranes and <0.2 μg of each recombinant polypeptide. Since binding of GMAP-210 to membranes was shown to resist 0.5 M NaCl we performed the binding assay in the presence of this salt concentration. After sedimentation of Golgi membranes, binding of the recombinant polypeptides was detected by IB using the AS. In the absence of Golgi membranes, any recombinant polypeptide sedimented. Plasmid DNA used for transfection was purified through two preparative CsCl/ethidium bromide equilibrium gradients, followed by phenol extraction and ethanol precipitation. COS cells were split 24 h before transfection so that they were 60–80% confluent on the day of transfection. 2–5 × 10 6 cells/assay were resuspended in 200 μl of 15 mM Hepes-buffered serum-containing medium, mixed with 50 μl 210 mM NaCl containing 5–20 μg plasmid DNA and electroporated using a Bio-Rad Gene Pulser. 6 h after electroporation, medium was replaced by fresh medium and cells were processed for immunofluorescence (IF) staining after 24–48 h. Indirect IF was carried out as described . For GFP-fluorescence, cells were fixed in −30°C methanol for 3 min. The samples were observed either using a Leica microscope and a Hamamatsu chilled CCD camera or using a TCS4D confocal laser scanning microscope based on a DM microscope interfaced with an Argon/Krypton laser. Simultaneous double fluorescence acquisitions were performed using the 488-nm and the 568-nm laser lines to excite FITC and TRITC dyes using a 63× or 100× oil immersion PlanApo objectives. Images were transferred to a Macintosh computer for editing. All documents presented except Fig. 8 (CCD images) are two-dimensional projections of images collected at all relevant z-axes. Microtubules were assembled in vitro from phosphocellulose-purified bovine brain tubulin as described above. To generate more polymer ends while keeping the polymer mass constant, microtubules were sheared by repetitively passing them through a 26-gauge hypodermic needle attached to 1-ml insulin syringe. HeLa cells were incubated with 20 μM taxol for 4 h at 37°C and then extracted with 1% NP-40 in PEM buffer. NP-40 soluble fraction from taxol-treated cells (fraction TS) contained a pool of GMAP-210 and γ-tubulin but no α-tubulin as revealed by IB . Identical amounts of TS fraction were mixed with equal amounts of microtubules before (−) or after (+) shearing, incubated for 30 min at room temperature and then centrifuged through a 10% sucrose cushion at 60,000 g for 30 min at 4°C. TS fraction and microtubule pellets were analyzed by IB for the presence of GMAP-210, α-tubulin and γ-tubulin. cDNA clones for GMAP-210 were obtained after immunoscreening of a HeLa cDNA library with the AS (clones R1–R6 and R8) and rescreening with an R8 fragment as a probe (clone R9). Clones were routinely expressed in fusion with GST and analyzed by IB using affinity-purified anti–GMAP-210 antibodies. Sequence analysis revealed that the 3′ end was missing but a sequence database search detected several expressed sequence tags whose 5′ ends were identical to the 3′ end of R9. Clone RT-10, containing the stop codon and the polyadenylation signal, was obtained by RT-PCR using primers P1 and P2 . This clone showed a very weak positive reaction in blots revealed with affinity-purified anti–GMAP-210 antibodies, explaining why the 3′ end of the protein was not obtained by screening of the cDNA library with the AS. The combined sequence derived from the overlapping clones R2, R3, R5, R6, and RT-10 comprises a total of 6452 bp and contains an open reading frame of 5,937 bp. The predicted protein sequence consists of 1,979 amino acid residues and has a calculated molecular mass of 223,902 and a predicted pI of 5.1, consistent with the molecular weight and pI of GMAP-210 determined by one- and two-dimensional gel electrophoresis . The PROSITE Dictionary of Proteins Sites and Patterns revealed multiple consensus motifs for phosphorylation and two leucine zipper patterns between amino acids 413–448 and 1,440–1,461. No known consensus nucleotide-binding motifs were found consistent with the lack of a direct effect of ATP or GTP on the binding of GMAP-210 to microtubules. Analysis of GMAP-210 amino acid sequence with PepCoil program showed that a significant portion of the protein had a high probability of forming an extended coil . The secondary structure and charge display of GMAP-210 was predicted using the programs PeptideStructure and PepStats. According to this analysis, GMAP-210 appears as a very long coil encompassing most of the sequence although interrupted by a 100–amino acid stretch poorly structured and slightly basic. The NH 2 terminus is highly acidic and contains a small coiled-coil domain whereas the COOH terminus is a highly basic proline and glycine-rich domain . The two longest coils are interrupted by two short segments that contain proline residues. Since proline usually disrupts an α-helix, GMAP-210 seems to be organized into five coiled-coil segments that are separated by non-helical linkers . The presence of two leucine-zippers each localized in a long coiled-coil domain, further supports the possibility of an oligomeric form for GMAP-210 in vivo. Finally, the high content in proline and glycine (19%) in the basic COOH-terminal domain suggests a globular structure for this portion of the protein. Potential protein sequence similarities between GMAP-210 and known proteins were analyzed by searching the GenBank and SwissProt data libraries using tFASTA and BLAST. 20% identity of amino acid sequence was found between GMAP-210 sequence predicted to be coiled-coil and several structural and motor proteins of the cytoskeleton that contain coiled-coil domains such as CENP-E and CENP-F, NuMA, CLIP-170, myosin heavy chains, kinesins and dyneins, and the yeast proteins Nuf1, Uso1p, and Cut14. Comparison of GMAP-210 sequence with those of previously reported Golgi autoantigens such as p230 (golgin 245), GM130 (golgin 95), or GCP170 (golgin 160) revealed no significant similarity (18% through coiled-coil regions). The tissue expression pattern of GMAP-210 mRNA was studied by Northern blot analysis using two clones as probes, corresponding to 5′ and 3′ ends. Both probes revealed a single transcript of >7 kb that was more abundant in testis, heart, placenta, skeletal muscle and pancreas although it could be also detected in other tissues (not shown). The size of revealed mRNA easily exceeds that required to encode a protein of 223 kD, supporting that the isolated cDNA codes for GMAP-210. To definitively demonstrate that the cDNA sequence corresponded to that of the p210 autoantigen, we have generated two polyclonal antibodies against two GST-fusion proteins containing distinct regions of GMAP-210 . These polyclonal antibodies, named RM127 and RM130, revealed by IB a band of identical electrophoretic mobility as that recognized by the AS and decorated the Golgi apparatus by IF . Most importantly, GMAP-210 was immunoprecipitated from PNS fractions of HeLa cells using the AS, RM130, and RM127 antibodies and immunoprecipitates were further analyzed by IB using RM130 polyclonal antibody. As expected GMAP-210 was detected in all cases , clearly demonstrating that the isolated cDNA encodes GMAP-210. The first indication that GMAP-210 could be a microtubule-binding protein came from its behavior during cell treatment with microtubule-active drugs . In untreated HeLa cells, GMAP-210 distributed in both Triton X-100 soluble and insoluble fractions matching the distribution of tubulin in these fractions. In taxol-treated cells, all tubulin became insoluble whereas it was still possible to detect part of GMAP-210 in the soluble fraction. Finally, prolonged treatment of HeLa cells with NZ induced complete depolymerization of tubulin and concomitantly complete solubilization of GMAP-210. As a control, distribution of actin was analyzed and as expected, remained unchanged under all of these treatments. The association of GMAP-210 with microtubules was further analyzed in vitro. We investigated whether GMAP- 210 cosedimented with microtubules purified from HeLa cells using taxol. A post-nuclear supernatant was prepared and centrifuged at high speed in order to obtain a supernatant depleted of most of membrane organelles. This high speed supernatant (HSS), that contained a pool of GMAP-210 , was incubated with taxol to polymerize endogenous tubulin. Microtubules were further purified by sedimentation through a sucrose cushion. GMAP-210 cosedimented with microtubules as revealed by IB using the AS . When HeLa cytosol was incubated instead with NZ to prevent tubulin polymerization, neither tubulin nor GMAP-210 sedimented through the sucrose cushion, indicating that GMAP-210 sedimentation was microtubule-dependent (not shown). To investigate the microtubule-binding characteristics of GMAP-210, taxol-polymerized microtubules were incubated with 0.5 M NaCl, 10 mM ATP, or 10 mM GTP, centrifuged through sucrose, and then the supernatants and pellets analyzed for the presence of GMAP-210 by IB . Under all these conditions GMAP-210 was detected in the microtubule-containing pellet. Similar results were obtained when these agents were added to cytosol before incubation with taxol in order to prevent the association (not shown). Only when 2 M urea was added to taxol-polymerized microtubules, could GMAP-210 be detected in the supernatant but, in these conditions, approximately half of tubulin was unpolymerized . From these experiments, we conclude that GMAP-210 binds tightly to microtubules in vitro, in a nucleotide-insensitive manner. To determine whether GMAP-210 binding to microtubules is direct and to decide which domain(s) is responsible for this interaction, a panel of GST-fusion proteins containing different portions of GMAP-210 or the complete protein was prepared and immobilized on glutathione-agarose beads. The immobilized proteins were allowed to interact with microtubules assembled from bovine brain phosphocellulose-tubulin with taxol. The presence of microtubules bound to wild-type or mutant proteins was detected by IB using an anti–α-tubulin antibody . These experiments demonstrated a direct binding of GMAP-210 to microtubules and further established that the microtubule binding site(s) corresponded to the region containing amino acids 1,712–1,979, the basic COOH-terminal domain. A similar experiment was then carried out but endogenous GMAP-210 was used instead of recombinant GST– GMAP-210 fusion protein. GMAP-210 was immunoprecipitated from HeLa cell lysates using AS, RM127, or RM130 antibodies precoupled to Sepharose beads. Beads were then incubated with microtubules assembled from bovine phosphocellulose-tubulin with taxol. Fig. 4 B shows that endogenous GMAP-210 efficiently interacted with microtubules. In the absence of added microtubules, no tubulin was detected (RM130-Mts) indicating that GMAP-210 did not bind soluble tubulin present in the cell lysates. To further characterize the association of GMAP-210 with microtubules in situ, we analyzed the microtubular cytoskeleton by selective extraction of cells . To do so, untreated (Control), 10 μM NZ-, or 10 μM taxol-treated cells were permeabilized with a non-ionic detergent in order to extract cytoplasmic proteins (fraction S). Then, microtubules were depolymerized and extracted with a cold-calcium–containing buffer (fraction Ca). All fractions obtained together with the detergent- and calcium-insoluble fractions (fraction I) were processed by IB to analyze the distribution of tubulin and GMAP-210. Fig. 5 A shows that, unexpectedly, tubulin was present in detergent- and calcium-insoluble fractions corresponding to control and taxol-treated cells. This result suggested that a subpopulation of microtubules could resist the depolymerization induced by cold-calcium. To confirm this assumption, untreated-, NZ-, and taxol-treated cells were extracted with NP-40 and cold-calcium. Cell ghosts were then processed for IF and labeled with an anti–α-tubulin monoclonal antibody . According to biochemical data, some microtubules growing from the centrosome could be observed in untreated cells and no microtubules were present in NZ-treated cells whereas many microtubules resisted extraction in taxol-treated cells. GMAP-210 was absent from all Ca-soluble fractions but present in insoluble fractions corresponding to control and taxol-treated cells . This result indicates that GMAP-210 does not bind to all assembled microtubules in situ but interacts preferentially with a subset of cold-calcium–resistant microtubules. GMAP-210 was also detected in the soluble fractions under all conditions suggesting that binding of GMAP-210 to microtubules in vivo must be regulated since all GMAP-210 did not bind to microtubules even when all tubulin became polymerized with taxol . To characterize this subpopulation of stable microtubules, we quantitatively analyzed the distribution of detyrosinated α-tubulin (glu-tubulin) with respect to total tubulin (α-tubulin) in the three fractions obtained from control cells, using the same subfractionation procedure . In untreated cells, tubulin was found to distribute as follows: 35% was soluble, 52% was polymerized in microtubules that were sensible to cold-calcium (corresponding to 80% of total microtubules), and 13% in microtubules resistant to these conditions (corresponding to 20% of microtubules). This subpopulation of more stable microtubules contained 29% of the total detyrosinated α-tubulin whereas 80% of more labile microtubules contained 67% of glu-tubulin. These results indicate that resistant microtubules contain 1.8 times more glu-tubulin than the rest. Thus, GMAP-210 appears preferentially associated in situ to a subpopulation of stable microtubules that are enriched in detyrosinated α-tubulin. The binding site for Golgi membranes on GMAP-210 was also mapped by deletion analysis. A series of NH 2 -terminal and COOH-terminal truncation mutants were constructed and expressed as GST-fusion proteins . They were immobilized on glutathione-agarose beads and further treated with thrombin to release recombinant proteins from GST. Golgi membranes were purified by flotation in a sucrose gradient, washed with 2 M NaCl to remove endogenous GMAP-210 and centrifuged at high speed. 10 μg of washed Golgi membranes were incubated with <0.2 μg of different fragments of GMAP-210 in the presence of 0.5 M NaCl. After centrifugation, both supernatants and pellets were analyzed by IB using the AS . The constructs ΔC611 and ΔC375 appeared enriched in Golgi membranes containing pellets, whereas fragments lacking NH 2 -terminal domain mostly remained in supernatants. Binding of GMAP-210 NH 2 terminus to Golgi membranes was further confirmed in vivo by transfection experiments (see below). To approach the function of GMAP-210 in the organization of the Golgi apparatus, we transiently expressed intact or mutant proteins in COS cells. Full-length cDNA coding for GMAP-210 with or without an NH 2 -terminal HA-epitope was introduced in cells by electroporation. Cells were incubated, fixed, double labeled and finally observed in a confocal laser microscope. An effect on the structure of the Golgi apparatus and microtubule network could be observed depending on the expression level of the transfected protein. At low expression level, the full-length GMAP-210 localized to a compact juxtanuclear reticulum characteristic of the Golgi apparatus as revealed by an anti-HA antibody (not shown). The labeling pattern overlapped exactly with that exhibited by the AS that recognized both exogenous and endogenous proteins, indicating that the expressed protein is targeted to the same place as the endogenous GMAP-210, namely the CGN. No significant changes in the structure of the Golgi complex or the microtubule network could be appreciated (not shown). By contrast, overexpression of GMAP-210 dramatically perturbed the organization of the Golgi apparatus. Under these conditions, transfected cells could be easily distinguished from the surrounding nontransfected cells by IF, due to the high amount of exogenous GMAP-210. Several features could be observed. First, a dramatic enlargement of the Golgi apparatus that could remain as a single pericentrosomal structure or as multiple fragments centrosomally localized but also distributed throughout the cytoplasm . Double labeling for GMAP-210 and the medial Golgi marker CTR433 or the cis-Golgi marker p115 revealed that not only the CGN but also the other compartments of the Golgi apparatus were distorted by overexpression of GMAP-210. Remarkably, the spatial distribution of GMAP-210 and the Golgi markers was largely coincident, although they demonstrated a graded inverse distribution: GMAP-210 labeling increased towards the periphery whereas the intensity of CTR433 and p115 decreased. A careful analysis of optical cuts suggested an hypertrophy of the CGN extending towards the cell periphery whereas the remnant of the Golgi stacks remained close to the nucleus . Second, the array of microtubules in the centrosomal area appeared modified in overexpressing cells. The microtubule aster became undefined and many microtubules appeared to emanate from the enlarged Golgi area rather than from the centrosome . In overexpressing cells in which the Golgi complex was fragmented, perturbation was more pronounced and microtubules appeared randomly distributed. We further showed that the Golgi perturbation provoked by GMAP-210 overexpression depended upon the microtubule-binding capacity of the protein: when a truncated form of GMAP-210 lacking the COOH-terminal microtubule-binding domain was overexpressed, it clearly accumulated at the Golgi apparatus although an homogeneous cytoplasmic staining was also observed . Comparison between a ΔC1778 overexpressing cell and a nontransfected cell revealed no significant differences in the Golgi apparatus or the microtubule network structure. It must be noted that overexpression of this mutant had no detectable effect on the distribution of endogenous GMAP-210. We also studied the effect of microtubule-active drugs on the phenotype displayed by GMAP-210 overexpressing cells. When transfected cells were incubated with NZ, the Golgi apparatus became fragmented and scattered throughout the cytoplasm in all overexpressing cells and complete colocalization with CTR433 and p115 was observed (not shown). Transfected cells were also incubated with taxol to promote the formation of microtubule bundles. In the presence of taxol, GMAP-210–enriched membranes preferentially redistributed at the periphery of the cells, whereas the rest of the Golgi complex remained juxtanuclear . GMAP-210–enriched membranes were found to be always located at one end of microtubule bundles . Higher magnifications at the cellular periphery allowed to distinguish individual microtubules and GMAP-210–enriched membranes could be seen associated with them . Thus, taxol-induced microtubules were able to tear the GMAP-210–enriched elements away from the rest of the Golgi apparatus. To further characterize the functional domains of GMAP-210, both NH 2 -terminal and COOH-terminal domains were cloned in fusion with the green fluorescent protein and transiently expressed in COS cells. ΔC375-GFP mutant appeared mostly cytosolic but also localized in punctate structures clustered in the Golgi region . Labeling with an antibody that specifically recognized endogenous GMAP-210 revealed partial colocalization . This result indicated that the truncated chimera was able to bind to Golgi membranes. At higher expression level, endogenous GMAP-210 was hardly visible consistent with the truncated mutant displacing the endogenous protein from membranes . Under these conditions, the medial Golgi marker CTR433 also showed an altered distribution . Instead of the normal compact Golgi seen in the untransfected cell in this field, CTR433 appeared in some punctate structures in the Golgi area and the labeling intensity had considerably decreased. Therefore, Golgi compartments other than the CGN appeared morphologically perturbed in the absence of membrane-bound GMAP-210. These data together with those presented in Fig. 6 demonstrate that the NH 2 terminus of GMAP-210 is involved in targeting the protein to Golgi membranes and strongly suggest that GMAP-210 is required for processes involved in maintaining the integrity of the Golgi apparatus. Similar experiments were carried out with the microtubule-binding domain for which GFP was placed in the NH 2 terminus of the mutant protein. Contrary to our expectations, the GFP-ΔN1778 mutant protein did not decorate microtubules. At low expression level, one or two spots that colocalized with the centrosomal marker CTR453 could be observed . With increasing expression levels of the mutant, increasing accumulation of GFP at the centrosome occurred together with the dispersion of the pericentriolar material that became almost undetectable with the monoclonal antibody CTR453 . Centrioles were present in the center of the GFP-labeled centrosomes, as revealed by anti-detyrosinated tubulin antibodies (not shown). After longer periods of expression, GFP-ΔN1778 accumulated massively at the centrosome although heterogeneous sized bright foci of GFP scattered throughout the cytoplasm were also observed. The Golgi apparatus was displaced to the periphery of the massive centrosomal aggregate that appeared surrounded by a dense network of detyrosinated microtubules . The finding that the COOH-terminal microtubule-binding domain of GMAP-210 localizes in vivo to centrosome strongly suggested that GMAP-210 might interact only with the minus ends of microtubules. One prediction of this hypothesis is that for constant polymer mass, GMAP-210 binding to microtubules in vitro will increase when more ends are present. To test this, we sheared a constant mass of microtubules (polymerized from phosphocellulose-bovine brain tubulin using taxol) by passing them through a hypodermic needle attached to 1-ml syringe. In parallel, we prepared a NP-40 soluble fraction from HeLa cells that had been treated with taxol for 4 h in order to completely polymerize the endogenous tubulin. This tubulin-free fraction (TS) contained a pool of GMAP-210 and γ-tubulin, as revealed by IB . Equal amounts of fraction TS were mixed with equal amounts of microtubules before (−) or after (+) shearing. After incubation, microtubules were centrifuged through a sucrose cushion and microtubule-pellets analyzed by IB. A typical experiment is shown in Fig. 12 (top) and the mean of values of three independent experiments is represented in Fig. 12 (bottom). Shearing increased the number of microtubules twice as determined by quantification of bound γ-tubulin before and after shearing. By using the same procedure Li and Joshi obtained an increase in the microtubules number of 1.5 times, fitting well with our results. The amount of GMAP-210 bound to microtubules also increased 1.8 times indicating that GMAP-210 interacts only with the ends of microtubules. Significant progress has been made towards understanding the molecular mechanisms of microtubule-dependent motors and their roles in membrane traffic. By contrast, the possible role of other microtubule-binding proteins lacking motor activity in the organization of the central membrane system has remained elusive. We report here the cloning and functional characterization of a previously described CGN-associated protein, p210. Several pieces of evidence argue for GMAP-210 being a microtubule-binding protein. First, the presence of GMAP- 210 in Triton-extracted cell ghosts is always dependent on the existence of polymerized microtubules. Second, GMAP-210 cosediments with taxol-polymerized microtubules from HeLa cells and remains associated in situ with stable microtubules. Finally, both endogenous GMAP-210 and purified bacterially expressed GMAP-210 interact with purified bovine brain microtubules. Interestingly, GMAP-210 binding to microtubules increases with the number of free ends in a given mass of microtubule polymers. This was demonstrated by shearing microtubules into short fragments, hence keeping the polymer mass constant. Interaction of GMAP-210 with polymerized tubulin appears to be a tight nucleotide-insensitive binding as it is not released from the microtubule pellet by salt conditions that are known to dissociate most MAPs, or by ATP or GTP addition. These binding characteristics correlate well with those reported for interaction between the Golgi complex and microtubules and differ from those exhibited by CLIP-170, motors, or other MAPs. The complete sequence of GMAP-210 has been obtained . Recently, an almost identical sequence, submitted to data banks after submission of our sequence, has been reported under the accession number AF007217 . This protein was reported to be a thyroid hormone receptor coactivator, negatively regulated by the retinoblastoma protein and named Trip230. We have no explanation for such a functional discrepancy but we are confident that the sequence we have cloned corresponds to the p210 autoantigen associated to the Golgi apparatus described previously by all the experimental data presented in this paper. In addition, a partial cDNA sequence of a novel gene named CEV-14 that corresponds to amino acids 1,188–1,753 of GMAP-210 has been reported. This partial sequence has been identified as the NH 2 -terminal half of a fusion gene in which the COOH-terminal half is a fragment of the platelet-derived growth factor receptor containing transmembrane and tyrosine kinase domains. CEV14-PDGFR fusion gene is generated by a chromosomal translocation that is associated with acute myelogenous leukemia . Our finding that this fragment corresponds to the second large coiled-coil domain of GMAP-210 supports the idea proposed by the authors that the mechanism of transformation is the ligand-independent dimerization of PDGFR leading to ectopic constitutive tyrosine kinase activation. Structural features predicted by the molecular characterization of GMAP-210 indicate a long α-helical domain with high potential to form a coiled-coil structure. Such a long coiled-coil domain has been found in other autoantigens associated with the cytoplasmic face of the Golgi membrane including giantin or GCP372 , golgin–245 or p230 , golgin–95 or GM130 , and golgin 160 or GCP170 . GMAP-210 is distinct from previously described microtubule-interacting proteins. GMAP-210 is predicted to possess a three domain structure consisting of the long α-helical central domain and the nonhelical end domains, similar to other proteins that interact with the cytoskeleton. The organization of charged regions of GMAP-210 is also reminiscent of that of several other MAPs. The NH 2 -terminal end is acidic whereas the COOH-terminal domain has a high pI like MAP2 , MAP4 , tau , or NuMA . However, it is inverse to CLIP-170 that contains a basic NH 2 -terminal domain, a central coiled-coil domain, and an acidic COOH-terminal domain. The highly basic COOH-terminal region of GMAP-210 was shown to mediate direct interaction with microtubules. Neither the acidic NH 2 -end, nor the long α-helical domain associate with microtubules in vitro. Thus, GMAP-210 behaves like most MAPs characterized so far, in which basic regions play a significant part in conferring ability to bind microtubules. The microtubule-binding domain of GMAP-210 shows no overall homology to any of previously characterized sites. It does not contain internal repeats like MAP2, tau, or CLIP-170, suggesting that only one binding site is present in each GMAP-210 molecule. The binding domain of GMAP-210 to the cytoplasmic side of CGN membranes has been localized in the NH 2 terminus of GMAP-210. This region consists of two acidic nonhelical regions interrupted by a small coiled-coil domain and followed by a large coil. The acidic COOH-terminal domain of CLIP-170 has also been postulated to interact with other proteins on the surface of endocytic organelles. A molecular design consisting of an elongated molecule containing a conserved microtubule-binding region at one end and a variable domain at the other end involved in specifying interactions with organelles has been proposed for nonmotor proteins implicated in linking cytoplasmic organelles to microtubules such as CLIP-170 . Although no significant sequence homology with CLIP-170 out of the coiled-coil region has been found, the molecular design of GMAP-210 fits well with this model. The biochemical data provide strong evidence that GMAP- 210 is a Golgi-associated protein that directly interacts with microtubule ends. Consistent with this, overexpression of GMAP-210 induced perturbations in both the Golgi apparatus and the microtubule network, and these effects increased with the expression level of the protein. Exogenous GMAP-210 was correctly addressed to the Golgi apparatus that appeared dramatically enlarged and sometimes fragmented in structures scattered throughout the cytoplasm. This hypertrophy of the Golgi apparatus occurred together with a modification of the microtubule network that mainly affected the Golgi-centrosome area. In fact, transfected cells could be easily identified by looking at the microtubule network, due to the absence of a distinct microtubule aster at the centrosome. Instead, a dense network of short microtubules could be appreciated colocalizing with the enlarged Golgi apparatus, and cytoplasmic microtubules seemed to emanate from the periphery of this perturbed region. Deletion of the COOH-terminal domain completely abolished both effects, strongly suggesting that the basic COOH terminus is responsible for binding of GMAP-210 to microtubules in vivo as well as in vitro. These data also indicate that the perturbation of the Golgi apparatus produced by GMAP-210 overexpression is primarily due to an excess of binding to microtubules. It was intriguing that truncated GMAP-210 lacking microtubule-binding domain accumulated mostly in the cytosol whereas intact GMAP-210 associated almost completely with Golgi membranes. The ΔC1778 mutant partially associated with Golgi membranes without apparently displacing endogenous GMAP-210, suggesting that endogenous GMAP-210 is present at subsaturating levels in cells and that interaction of GMAP-210 with membranes is saturable. This view is also consistent with the results obtained in vitro in which purified GMAP-210–containing membranes were able to bind a limited amount of ΔC611 or ΔC375 mutants. But how to understand that GMAP-210 can steadily accumulate on CGN membranes in cells overexpressing the full-length protein if the interaction of GMAP-210 with CGN membranes is saturable? An amplification mechanism could reasonably explain this paradoxical observation: the progressive accumulation of GMAP-210 to the CGN would induce the stabilization of microtubules in this area that would allow the progressive enlargement of CGN elements. Such an increase in membrane surface would in turn allow that more GMAP-210 accumulates. Numerous observations indicate that the size of the Golgi apparatus is maintained by a fine tuning of the membrane flux in and out of the Golgi complex. Overexpression of GMAP-210 produces a significant increase in the Golgi apparatus size. On the contrary, when endogenous GMAP-210 is displaced from Golgi membranes by overexpression of a truncated mutant containing only the membrane binding domain, the size of the Golgi complex decreased . Thus, GMAP-210 seems to play a role in determining the size of the Golgi apparatus, probably by regulating the number of microtubule-binding sites on the surface of the CGN. A tighter association of the CGN with microtubules could prevent its maturation into Golgi cisternae or impede anterograde vesicular transport from the CGN to the medial Golgi , producing the enlargement of the CGN by accumulation of newly synthesized or recycling proteins. This could explain the partial colocalization of cis- and medial-Golgi markers with GMAP-210 in the enlarged CGN. At higher expression levels, translocation of pre-Golgi elements from peripheral ER sites to the Golgi area could be also affected and fragmentation of the enlarged Golgi apparatus would occur. Consistent with this, microtubules appeared randomly nucleated in cells in which the Golgi apparatus was fragmented. The relationship between CGN and microtubules thus appears as a finely regulated interaction in order to ensure not only the localization but also the steady state structure and size of the Golgi apparatus. Many recent studies have emphasized the role of microtubule motors not only in membrane traffic but also in determining the localization and morphology of the Golgi apparatus. It has been proposed that in the steady state the Golgi complex is in dynamic equilibrium between the anterograde force mediated by kinesin family motors and the retrograde force mediated by cytoplasmic dyneins rather than simply being associated with microtubules near their minus end . Our results indicate that association of CGN membranes with the minus end of microtubules is also necessary for the proper localization, morphology and size of the Golgi apparatus. They are consistent with a model in which dynein is responsible for moving Golgi elements towards the centrosome whereas GMAP-210 is regulating the static binding of the Golgi membranes to microtubules. We present here several lines of evidence showing that GMAP-210 is a minus end microtubule-binding protein. First, GMAP-210 binding to microtubules in vitro is related to the number rather than the mass of microtubule polymers. Second, the COOH-terminal domain of GMAP-210, responsible for the microtubule-binding property of the protein in vitro, localizes in vivo to the centrosome, where the minus ends of microtubules accumulate. Moreover, it is able to disorganize the pericentriolar material suggesting that it could displace other centrosomal proteins from the minus ends of microtubules. In the presence of NZ, GMAP-210 does not remain associated with the centrosome . Taxol treatment of GMAP-210 overexpressing cells induced specific redistribution of GMAP-210 enriched elements to the cell periphery where they associated with one end of microtubule bundles. Finally, in GMAP-210 overexpressing cells, a dense network of short microtubules appears in the Golgi area from which cytoplasmic microtubules seem to emanate, consistent with the notion that binding or capping of the minus ends of microtubules by proteins result in their stabilization. All data presented here support a model in which GMAP-210 serves to maintain the structural integrity of the Golgi apparatus by interacting with the minus end of microtubules. The NH 2 -terminal domain binds to CGN membranes whereas the COOH-terminal domain interacts with the minus end of microtubules at the centrosome. Remarkably, a low expression level of the COOH-terminal domain leads to the centrosomal localization of GFP in a manner identical to the centrosomal decoration obtained with an antibody directed against γ-tubulin , or against a γ-tubulin binding protein , i.e., the centriole pair and a domain surrounding it. From the study of γ-tubulin distribution in epithelial cells, Mogensen et al. have proposed that peripheral docking elements distinct from nucleating elements could be closely associated with centrosomes. In their model, microtubule minus ends would be capped and stabilized before being released from nucleating elements, which would remain in the juxtacentriolar vicinity to nucleate new microtubules. Control mechanisms would ensure that a microtubule is anchored to the centrosome periphery or released. Several reports have indeed suggested that centrosomally nucleated microtubules can escape to other locations, particularly in neurons but also in other cell types . Based on this idea, one could propose that free microtubule ends could be captured by GMAP-210 via its COOH-terminal domain. This might also account for the existence of a subpopulation of short, stable microtubules that colocalizes with the Golgi apparatus . This subpopulation is rich in detyrosinated microtubules and we have shown that GMAP-210 is specifically enriched in a microtubule fraction with similar characteristics. In this view, CGN membranes, rather than the centrosome itself, would be the anchoring sites for microtubules participating in Golgi dynamics and stability. A direct homologue of GMAP-210 has not been found in the yeast genome database. This is consistent with the fact that in lower eukaryotic cells no spatial segregation between the ER and the Golgi apparatus exists. In such cells, the Golgi complex is often found next to the ER sites and pre-Golgi elements form the first cisternae of the Golgi stack. However, in many eukaryotic cells the Golgi apparatus is centered around the centrosome and actively maintained there. Our data uncover a critical role for the CGN in this process and identify GMAP-210 as an important factor in mediating both the localization and the integrity of this Golgi compartment. During mitosis, the microtubule dynamics changes and the centrosomal localization of the Golgi apparatus is lost. One can speculate that GMAP-210 could be a target for the mitotic regulation of the CGN. Since GMAP-210 remains bound to Golgi membranes during mitosis (unpublished results), the obvious candidate for this mitotic regulation is the microtubule-binding domain. It is interesting to note in this regard that the COOH-terminal domain has two putative cyclin-dependent kinase phosphorylation sites. Experiments are now in progress to test this hypothesis. | Study | biomedical | en | 0.999996 |
10189371 | Pre-B leukemia cell line ALL-697 containing a mock vector (neo) or a Bcl-2 overexpression vector (Bcl-2) were kindly provided by Dr. John Reed (Burnham Institute, La Jolla, CA). T cell leukemia Molt-4 cells stably transfected with the vector carrying a hygromycin-resistant cDNA or a murine Bcl-2 overexpression vector were also used in this study . Molt-4 and ALL-697 cell lines were maintained in RPMI 1640 medium supplemented with 2.5% Hepes buffer and 10% fetal bovine serum ( GIBCO BRL ). All experiments were conducted under serum-free conditions. Vincristine (VCR) and etoposide were purchased from Sigma Chemical Co. [ 3 H]Arachidonic acid and [ 3 H]palmitic acid were purchased from NEN™ Life Science Products. Cells were seeded at 1.2 × 10 6 cells/ml in serum-free medium and labeled for 16 h with [ 3 H]-labeled free fatty acid (1.5 μCi/ml). The cells were washed twice with sterile PBS ( Sigma Chemical Co. ), seeded at 5 × 10 5 cells/ml in serum-free medium, and treated under various conditions as indicated in the text. The cells were collected in a microtube afterward and resuspended evenly. Sample of 200 μl was counted and labeled as total counts (A). The remaining cells in the microtube were pelleted at 2,000 g in a micro centrifuge for 5 min. The supernatant (200 μl) was transferred to another scintillation vial, and the radioactivity was counted as the amount of radioactivity release into the medium and termed B. The amount of release of radioactivity was expressed as percentage of B over A. Cells were seeded at 5 × 10 5 /ml in serum-free medium. Cells of 1 × 10 6 were treated under conditions indicated in the text, and were collected and washed once with phosphate buffered saline (pH 7.4). These cells were resuspended in the sample buffer. The whole cell extracts were separated by 6% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% dry milk and detected by anti-PARP antibody in a 1:2,000 dilution (Transduction Laboratories). ALL-697 pre-B cell line with and without human Bcl-2 overexpression vector and Molt-4 T cells with and without murine Bcl-2 overexpression vectors were prepared for EM after 3, 6, 12, and 24 h of exposure to the selected apoptotic inducer. Controls were identical cells grown for the same length of time without the inducer. Cells were spun at the relatively high speed of 2,000 g for 5 min to pellet cell fragments as well as intact cells (one set of experiments was conducted using a low speed spin just sufficient to pellet the cells). Cell pellets were immediately fixed at room temperature in 3% glutaraldehyde tousimis, 0.2% tannic acid (Electron Microscopy Sciences) in 10 mM MOPS (3-[ N -Morpholino]propanesulfonic acid) buffered mammalian ringer, pH 7.2 for 30 min. After rinsing in buffered ringer and then in PO 4 buffer, cells were post fixed in 1% OsO 4 (100 mM K 3 PO 4 , 10 mM MgCl 2 , pH 6.1) ice-cold for 20 min. Cells were block-stained with 2% aqueous uranyl acetate for 30 min at room temperature after rinsing twice in water, and dehydrated in a graded ethanol series 50–100% and embedded in Araldite 506 . Sections were cut with a diamond knife on a Reichert OMU3 ultramicrotome and stained with KMnO 4 and lead and photographed on a Philips EM 420. Cells were seeded at 5 × 10 5 cells/ml in RPMI 1640 serum-free medium. After the indicated times of treatments under conditions, as indicated in the text, cells were resuspended in 10 μl PBS, diluted at 1:1 ratio with Trypan Blue solution (0.4%; Sigma Chemical Co. ). The dead and live cells were counted using a hemocytometer. To study the regulatory mechanism of chemotherapy-induced apoptosis, we took advantage of the membrane lipid release assay . Pre-B cell leukemia ALL-697 cells were prelabeled overnight with [ 3 H]palmitate and treated with etoposide (10 mM), alone or together with either 60 μM Z-Asp-Glu-Val-Asp-CH 2 F (DEVD; an irreversible inhibitor of caspase 3 and related caspases), or the same concentration of N-Tyr-Val-Ala-Asp-aldehyde (YVAD; a reversible inhibitor of caspase 1 and related caspases). Fig. 1 A demonstrates that etoposide induced release of [ 3 H]palmitate in the form of membrane lipid such that by 12 h there was more than twofold increase of release of radioactivity as compared with control cells. In contrast, in the presence of DEVD, the etoposide-induced release of radioactivity was inhibited throughout the time course evaluated, and remarkably, this inhibition continued up to 72 h of etoposide treatment. The inhibitor of caspase 1, YVAD, could not effectively inhibit etoposide-induced release of labeled membrane lipid in ALL-697 cells even at the earliest time point (12 h). We used both reversible and irreversible forms of DEVD and YVAD in this assay. Both forms of DEVD were inhibitory, whereas neither form of YVAD was inhibitory (data not shown). To establish the effective concentration for the caspase inhibitors, we conducted a dose response study using varying concentrations of either DEVD or YVAD on the release of membrane lipids, as an indicator of release of apoptotic bodies. DEVD or YVAD ranging from 1 μM to 100 μM were used. Fig. 1 B demonstrates that etoposide treatment (10 μM) induced release of membrane lipids within 6 h. The addition of YVAD up to 100 μM had no effect on the release of membrane lipids. In contrast, the addition of DEVD effectively inhibited the release of membrane lipids at a concentration as low as 10 μM. At 50 μM, the release of membrane lipid was completely inhibited . Thus, in the following experiments, we chose 60 μM as our effective concentration of caspase inhibitor on the regulation of apoptosis induced by chemotherapeutic agents. These results indicate that the release of membrane lipids in chemotherapeutic agent-induced apoptosis could be effectively inhibited by DEVD, the inhibitor of caspase 3. In an effort to understand the relationship between the release of labeled membrane lipids and cell death, we next evaluated cell viability by Trypan Blue exclusion analysis. ALL-697 cells transfected with Bcl-2 or with a vector carrying the neomycin gene (neo) were evaluated for viability in response to etoposide (10 mM). Fig. 2 demonstrates that cells overexpressing Bcl-2 no longer proliferated in the presence of etoposide, but these cells were alive even up to 72 h of etoposide treatment, as no dead cells were present upon Trypan Blue exclusion analysis. In contrast, cells transfected with vector control started to die as early as 6 h, and by 24 h all the cells were dead. On the other hand, despite the fact that DEVD blocked membrane lipid release, it was unable to prevent cell death. ALL-697 cells pretreated with DEVD (60 μM) and then with etoposide initially displayed a slower rate of cell death as measured by Trypan blue exclusion assay, but by 24 and 48 h cell death increased to the same level as the cells that had not been exposed to DEVD . To demonstrate that cell death in response to etoposide in the ALL-697 was indeed apoptotic and was mediated by caspases, we next evaluated cleavage of the caspase 3 substrate poly (ADP-ribose) polymerase (PARP). PARP was cleaved from the intact 116-kD protein into the expected 85-kD proteolytic fragment after treatment with etoposide at 12 h . As expected, cells overexpressing Bcl-2 were protected from etoposide-induced PARP cleavage, consistent with the lack of cell death seen in Fig. 2 . Similarly, the addition of DEVD almost completely protected cells from PARP cleavage by 12 h, and significantly protected cells from PARP cleavage by 48 h. Under the same condition, the cells were beginning to die by 12 h, and were completely dead by 48 h. These data confirm that the activation of caspase 3 and the related enzymes were significantly inhibited by DEVD, but DEVD only slightly delayed but did not protect from cell death. Since DEVD inhibited membrane lipid release, these results also imply that caspase 3 activity is required in the release of membrane lipids. Bcl-2 inhibits the process that initiates apoptotic body formation whereas DEVD, the inhibitor of caspase, arrests the subsequent maturation and release of apoptotic bodies in chemotherapeutic agent-induced apoptosis. To gain understanding of the processes involved in apoptotic body formation and release, we next used EM to identify the structures and materials associated with these processes. Using high speed centrifugation (10,000 g for 30 min), we pelleted all the components released into the medium from either control or etoposide-treated cells. Our previous studies showed that under high speed centrifugation, more than 80% of the radioactivity released from the apoptotic cells could be pelleted. Thin section EM on the 10,000 g pellet showed heterogeneous vesicles, membrane fragments, condensed chromatin, and small debris containing some recognizable portions of mitochondria and parts of other organelles . Hardly any of these components were seen in the high speed pellet from the control cells, or in Bcl-2 overexpressing cells treated with etoposide. These studies indicated that the released membrane lipid was probably incorporated into these heterogeneous cellular fragments. We next processed whole cells for EM in an effort to capture and visualize the several stages in the process of apoptotic body formation, and gain insight into the mechanism by which membrane lipids are released. ALL-697 and Molt-4 cells carrying an empty vector or a Bcl-2 overexpressing vector were fixed at a series of time points with and without exposure to several apoptotic inducers. After treatment, whole cells were centrifuged at 12,000 g for 5 min and the cell pellet was fixed. In one experiment, we used 2,000 g for 5 min (just sufficient to pellet the cells) and in all other experiments, we centrifuged cells at 12,000 g to ensure that as much of the material released from the cells as possible would be brought down with the whole cell pellet. Consistent with the [ 3 H]-membrane lipid release assay, the extracellular space around cells treated with either etoposide or vincristine showed grossly more debris, membrane profiles, and vesicles than the untreated cells. Cells undergoing apoptosis also showed membrane blebbing . The apoptotic cells were different from the untreated cells in the following aspects: (a) The cytoplasm of apoptotic cells contained heterogeneous vesicles in significantly greater abundance than the control cells. The vesicles were of varying sizes and of varying content; luminal profiles varied from empty to dense, amorphous to particulate. Frequently, the contents of vesicles appeared to be in the process of being released from the apoptotic cells . There was a class of lysosome-like vesicles that were not seen in the untreated cells . (b) At early time points in apoptotic cells, the mitochondria were not disrupted, but at later time points, they appeared to be in various stages of involution. In some cases, it was clear that mitochondria were being converted to mid-size vesicles containing an amorphous substance of medium density . (c) The release of vesicles and the formation of apoptotic bodies were evident. Some of these cells were clearly pinching off portions of themselves . These budding portions fit all the descriptions of apoptotic bodies that include cytoplasm, vesicles, and fragmented nucleus. At early time points after exposure to apoptotic inducers, the majority of the cells showed irregular shapes and the nuclei were polymorphic (lobulated). Although the nuclear chromatin was denser than control cells, complete condensation of chromatin was seen more frequently at later time points . By 6 h of exposure to 10 μM etoposide, many cells showed completely condensed chromatin with no visible sub-structure and the surrounding cytoplasm was pale and granular, without organelles . These cells were classic apoptotic cells. Cells overexpressing Bcl-2 responded differently to inducers of apoptosis from untreated cells . Consistent with the experiments on release of [ 3 H]-membrane lipids, there were no apoptotic bodies formed in the Bcl-2–transfected cells. The extracellular space around the treated Bcl-2 cells was clean, with no heterogeneous vesicles, organelle debris, or electron dense substances. There was no recognizable change in the morphology of the nucleus compared with the control cells, i.e., chromatin condensation was inhibited completely, consistent with previous reports . However, some large vesicles with dense contents that appeared similar to lysosomal vesicles, seen in apoptotic cells, also appeared in the Bcl-2 cells exposed to vincristine or etoposide . The overall morphology of Bcl-2 cells under vincristine and etoposide treatment was similar to that of untreated Bcl-2 cells and untreated control cells. The effects of the protease inhibitor DEVD on the morphology of treated cells were quite different from that of Bcl-2 overexpression. We treated the cells with DEVD alone or in combination with vincristine or etoposide. In cells treated with DEVD alone, the cells looked identical to the control cells (data not shown). When the cells were treated with vincristine or etoposide in the presence of DEVD , their morphology changed dramatically. The extracellular space around the cells treated with chemotherapeutic agent plus DEVD was much cleaner than the cells treated with chemotherapeutic agent alone. Also, there were no broken vesicles surrounding the cell membrane. The major features of a typical response were: (a) Many cells treated with vincristine and DEVD became shrunken and had dark, condensed cytoplasm. The cells showed membrane-bound finger-like projections. (b) Abundant accumulation of various vesicles crowded the condensed cytoplasm, suggesting that these vesicles were trapped inside the cells. (c) The caspase inhibitor DEVD did prevent complete condensation of chromatin into dense pools; chromatin in the nuclei remained recognizable as dark masses against a somewhat lighter background. There was no fragmented chromatin in the presence of DEVD. However, contrary to what was expected, nuclei and chromatin appeared more dense and compacted than normal . The great density and shrinkage of the DEVD cells and the crowding of the remaining cytoplasm with accumulations of various vesicles, and the lack of apoptotic bodies in the extracellular space suggested that cells were beginning the process of formation of apoptotic bodies. However, the inhibition of caspase activation apparently arrested the formation of apoptotic bodies in a intermediate stage . This “intermediate stage” of apoptosis distinguished itself from other stages of apoptotic body formation by the apparent intracellular accumulation of various vesicles in the condensed cytoplasm. Comparison of Figs. 4 B vs. 4 D and 5 B vs. 5 D, together with our studies on membrane lipid release suggested that the heterogeneous vesicles that appear to accumulate in the cytoplasm of the cells treated with DEVD and chemotherapeutic agents are possible precursors of apoptotic bodies normally released in the extracellular space. The regulatory role of DEVD on the formation of apoptotic bodies suggested to us that the formation of apoptotic bodies is a multi-staged process. Thus, we sequentially investigated the stages of apoptotic body formation by studying the cellular morphological changes at various time points (3, 6, and 24 h) in response to treatment of chemotherapeutic agents . The various stages of apoptotic body formation can be best illustrated in Fig. 6 A (stage 1 though stage 4). The early stage of apoptotic body formation is characterized by the initial condensation of chromatin . In this initial phase, the overall structure of chromatin is still recognizable, although compared with the normal nucleus , the chromatin is already somewhat condensed. The cell membrane is intact at this stage. The next stage shows the intermediate stage of apoptotic body formation, where various vesicles are formed and their contents are being released into the extracellular space, together with the striking condensation of chromatin. Clearly, the activation of caspases is required in the intermediate step between the formation of heterogeneous vesicles and the release of apoptotic bodies. The final stages of apoptotic body formation are illustrated in Fig. 6 A (labeled 3 and 4), where the cells appear as ghost structures in which all vesicles and apoptotic bodies have been released and the cytosol is uniformly granular and only a small dense pool indicates the former nucleus. At this stage, the extracellular space is littered with granular ghosts of cells that completed apoptosis at earlier times. In fact, at this late stage, the cells resembled those described by Wyllie and Kerr as “secondary necrosis,” probably since “in vitro” the dead cells are not absorbed by surrounding cells. These stages of apoptosis are also illustrated in the time course of treatment . Fig. 6 B shows the typical cell morphology after 3 h of etoposide treatment, as chromatin condensation has already started at this time (arrow). After 6 h of etoposide treatment, various vesicles have formed in the cytoplasm, combined with the striking condensation of chromatin . After 24 h of vincristine treatment or etoposide (data not shown), apoptotic cells have completed the release of apoptotic bodies, and the ghost structure of cells are the only remnants. An interesting observation is that cells that had entered mitosis appeared not to be affected by vincristine, since after 24 h of vincristine treatment, the only surviving cells were those that had entered mitosis . These surviving mitotic cells were numerous in vincristine-treated cells, but appeared far fewer in the presence of etoposide, possibly as a result of the effects of vincristine on G 2 /M progression. Comparing the response of cells overexpressing Bcl-2 to cells treated with DEVD suggested to us that multiple factors regulate the initiation, maturation, and release of apoptotic bodies. The overexpression of Bcl-2 totally inhibited the formation of apoptotic bodies. Compared with control cells, the Bcl-2 cells appeared normal even under electron microscopy after exposure to vincristine and etoposide for extended times. There was no accumulation of vesicles or fragmentation of chromatin in Bcl-2 cells in the presence of chemotherapeutic agents, as an early indicator of the formation of apoptotic bodies. More importantly, treated Bcl-2 cells maintained their viability for a long time. On the other hand, the inhibitor of caspases could not totally reverse the process of the formation of apoptotic bodies. However, DEVD interfered with the later stage of apoptotic body formation by inhibiting the release of apoptotic bodies, leading to the accumulation of various vesicles inside the cells and somewhat condensed chromatin. Therefore, the activation of caspases appears to be a necessary step in the subsequent maturation and release of apoptotic bodies. Overall, our studies demonstrate that formation of apoptotic bodies is a multi-staged process, characterized by the appearance of heterogeneous vesicles as the possible precursor of apoptotic bodies. Bcl-2 and the inhibitor of caspases differentially regulate the process of apoptotic body formation. Overexpression of Bcl-2 protein acts upstream of apoptotic body formation by completely inhibiting the initiation of this process, whereas the inhibition of caspase only inhibited the later stage of apoptotic body release. This study demonstrates that the formation of apoptotic bodies is a multi-staged process that is regulated differentially by Bcl-2 and caspases. Since the results clearly distinguish the action of Bcl-2 from that of caspases, they suggest that the effects of Bcl-2 transcend the regulation of caspases. In essence the results argue against proposed unitary pathways whereby Bcl-2 functions primarily to regulate caspases. This study reveals several differences between Bcl-2 and caspases in regulating apoptosis. First, Bcl-2 prevents the loss of viability in chemotherapy-induced apoptosis, but inhibitors of caspases do not. Second, Bcl-2 prevents most of the manifestation of apoptosis at the ultrastructural level, as shown by the near normal appearance of cells in response to chemotherapeutic agent-treatment whereas caspase inhibitors fail to inhibit many features of apoptosis, including cell volume loss and chromatin condensation. Third, Bcl-2 prevents the formation of apoptotic bodies completely, whereas caspase inhibitors do not interfere with the initial process that lead to apoptotic body formation, but DEVD clearly inhibits release of apoptotic bodies and vesicles. These results have several important implications in current research on apoptosis. EM studies reveal several phases of apoptosis and apoptotic body formation. Upon induction of apoptosis, the cells enter the initial phase of apoptotic body formation by the beginning of the condensation of chromatin. In this initial phase, the overall structure of chromatin is still recognizable, although compared with the normal nucleus, the chromatin is already somewhat condensed. The cells enter the intermediate stage of apoptotic body formation by forming heterogeneous vesicles in the cytoplasm and release these vesicles into the extracellular space. By this time, the chromatin is condensed to such an extent that no structure could be recognized. The nucleus is fragmented into several pieces. In the final stage of apoptosis, the cells appear as ghost structures in which all vesicles and apoptotic bodies have been released and the cytosol is uniformly granular and only a small dense pool indicates the former nucleus. This three-phase apoptotic process is regulated at least at two levels: Bcl-2 prevents the initiation of the first phase, whereas caspase inhibitors do not interfere with the initiation of the apoptotic program leading to the accumulation of precursor vesicles, but do prevent the release of apoptotic bodies from apoptotic cells . Our study also shows that Bcl-2, compared with DEVD, acts at a proximal step in the apoptotic pathway and that Bcl-2 can maintain cell viability, although it fails to maintain the cells' ability to proliferate. Therefore, Bcl-2 acts upstream of the “commitment site” in apoptosis. In contrast, activation of caspases in chemotherapy-induced apoptosis acts at a point downstream of the commitment step, as inhibition of caspase activity cannot prevent loss of cell viability. This divergence of the action of Bcl-2 and caspases raises interesting questions about the mechanism of action of Bcl-2 and its relation to caspases: although Bcl-2 has been studied extensively in apoptosis, the exact point at which Bcl-2 works to regulate the apoptotic program is still poorly defined. Bcl-2 has been found to interfere with the apoptotic pathway at a point upstream of caspases, inhibiting caspase activation upon induction of apoptosis . Activation of caspases could also be initiated by cytochrome c, another important regulator of apoptosis . Recent studies suggest that during apoptosis, cytochrome c is released from the mitochondria to the cytosol, and that the increased accumulation of cytochrome c in the cytosol triggers caspase activation. In addition, based on in vitro studies , it was shown that cytochrome c could trigger caspase activation directly by forming a protein complex with Apaf 1 (the human homologue of ced-4), and Apaf 3 . Overexpression of Bcl-2 has been shown to prevent the release of cytochrome c in apoptosis , implying that Bcl-2 acts upstream of the release of cytochrome c in apoptosis. Thus, it has been proposed that Bcl-2 acts primarily to inhibit the release of cytochrome c from mitochondria and the consequent activation of caspases . These and other results have led to the hypothesis that caspases are the primary executors of apoptosis and that Bcl-2 functions primarily to interfere with caspase activation. The results from our study argue against this simplified model for the regulation of apoptosis, at least in chemotherapy-induced apoptosis in lymphoid cells. Whereas Bcl-2 prevents chemotherapy-induced death, DEVD only inhibits a subset of the manifestations of apoptosis. Thus, Bcl-2 not only acts upstream of caspases, but must also act upstream of other non-caspase effectors of the apoptotic program. Our results are compatible with either of two scenarios: Bcl-2 may function primarily to inhibit cytochrome c release. In this case, the release of cytochrome c causes both activation of caspases required for release of apoptotic bodies and activation of other unknown effectors involved in initiation of the apoptotic program . Alternatively, Bcl-2 may inhibit a distinct pathway that activates early events in apoptosis in addition to inhibiting activation of caspases by cytochrome c . This study also raises some important considerations as to the role of caspases in chemotherapy-induced apoptosis: (a) caspase inhibitors do not prevent cell death in response to etoposide or vincristine, but they inhibit some of the manifestations of cell death such as PARP cleavage, the release of membrane lipids as the indication of pinching off apoptotic bodies and vesicle release, and partially inhibit the condensation of chromatin. This suggests that caspases are restricted to an execution role. (b) An initial regulatory role for caspases in chemotherapy-induced apoptosis is unlikely to be very significant, as once caspases are activated, the cells have already passed the commitment step in apoptosis. For example, inhibitors of caspases are not expected to ameliorate chemotherapy induced toxicity and side effects. While studying the role of caspases in apoptosis induced by Bak and DNA-damaging agents, McCarthy et al. showed that the caspase inhibitors ZVAD.fmk and BD.fmk could inhibit several aspects of cell death but could not inhibit membrane blebbing. The failure to inhibit membrane blebbing caused eventual cell death. In our studies, we have found that DEVD inhibits cellular blebbing effectively . It is possible that this difference is due to using different cells (they used Rat-1 fibroblasts where as we used leukemia cells). Therefore, we cannot further relate their observations to ours. Xiang et al. showed that in Bax-induced apoptosis, the treatment of protease inhibitors partially inhibited the condensation of chromatin and what they termed as “membrane vacuolation,” a process similar to what we describe as the intermediate stage of apoptotic body formation. All these studies however lend support to the notion that caspases have a role only in the execution phase of apoptosis, and after the commitment point has been passed in the signal transduction pathway in apoptosis. Finally, these observations show that care has to be exercised in interpreting specific assays in the evaluation of apoptosis. In chemotherapy-induced apoptosis in lymphoid cells, caspase inhibitors prevent PARP proteolysis, membrane release, and many nuclear changes, but they clearly do not inhibit ultimate cell death or changes in overall cell morphology and nuclear structure. Therefore, these observations underscore the importance of distinguishing the protection of cell viability from the inhibition of downstream events in apoptosis, such as chromatin condensation, and PARP cleavage. | Study | biomedical | en | 0.999996 |
10189372 | The COOH-terminal fusion construct of the cDNA for the MT-associated protein MAP2 with GFP cDNA has been described earlier . The fusion construct of the cDNA for mouse β6-tubulin isoform (kind gift of N. Cowan, New York University, New York) with the coding sequence of EGFP ( Clontech ) was constructed analogously into a beta-actin driven expression vector . Plasmids were purified using Qiagen columns. The rat embryo fibroblast cell line REF 52 was grown under standard conditions in DME supplemented with 10% FCS (Life Technologies). Cells were plated onto 18-mm round glass coverlips 14–20 h before transfection. Cultures were transfected using lipofectamine (Life Technologies) or Fugene 6 (Roche Diagnostics) according to the manufacturer's instructions. Both well-spread and rounded cells were manipulated. Well-spread cells came from cultures that had been transfected and plated some 24–72 h before experimentation. Rounded, spreading cells were obtained by replating cells that had been transfected 24–72 h earlier and manipulating them 2–6 h after replating. To induce retraction of cell edges in well-spread cells, cells were incubated on the microscope stage in Ca- and Mg-free PBS supplemented with 0.1 or 0.5 mM EDTA to chelate extracellular Ca and Mg ions resulting in a rounding of cells within 10–30 min. For live imaging, coverslips were mounted in observation chambers (Type 1; Life Imaging Services) in a special formula of DME with 1/10 of regular riboflavin content (Life Technologies) and imaged at 37°C on a Leica DM-IRBE inverted fluorescence microscope equipped with high numerical aperture oil lenses (Leica) and a GFP-optimized filter set (Chroma Technology). Images were captured using either a Kappa CF8/1 DXC (Kappa) or a MicroMax ( Princeton Instruments ) cooled CCD camera and MetaMorph Imaging Software ( Universal Imaging Corporation ). Figures were assembled with Adobe Photoshop and Illustrator. Cells were deformed by poking and prodding them with glass needles that had been calibrated to determine their bending constant, i.e., their resistance to deflection. The fabrication and calibration of needles has been described in detail and they have been used routinely to apply tension to cultured neurons . In brief, two needles were mounted in a micromanipulator; one needle was calibrated for its bending constant and used as the needle applied to the cell, while the other needle was used as an unloaded reference for bending of the calibrated needle and for possible drift of the micromanipulator system. The bending constants of the calibrated needles were between 10–30 μdyne/μm and some needles were pretreated with 0.1% polylysine and/or 50 μg/ml laminin to promote adhesion. Because of the high-magnification, high-NA objectives used to visualize GFP-tagged proteins in the cell and the high forces often applied to or exerted by REF 52 cells, it was not possible to keep both calibrated and reference needle within the digitized image frame captured by the computerized microscopy system. Instead, the bending of the calibrated needle was measured in real time from its deflection distance (relative to the reference needle) using an ocular reticle that had been calibrated with a stage micrometer. The nuclei of REF cells as well the actin cytoskeleton show almost purely elastic behavior in response to all manipulations that deformed their arrangement or shape. Fig. 1 shows an example in which the manipulating needle was poked into the nuclear region of a REF cell transfected with GFP–γ-actin, causing a sharp deformation of the nucleus and an accumulation of perinuclear actin at the tip of the needle. Actin not in the path of the needle showed no significant change in organization. After holding the deformation for ∼1 min, the needle was released (01:00). In this and other examples the nucleus behaved as a viscoelastic solid with an initial rapid phase of elastic recovery of most of its original shape (01:00 to 01:34) followed by a slower, apparently damped approach to original shape (02:52). Indeed, this behavior is qualitatively similar to a spring-and-dashpot model for neurite elasticity of cultured neurons . Nevertheless, we observed some net movement of actin and nucleus. For example, the arrows in Fig. 1 mark the initial position of the nucleus. As can be seen, the nucleus and its surrounding actin halo shifted somewhat in the direction of the experimentally applied force. As shown in Fig. 1 , we routinely observed that the nucleus and its surrounding GFP-actin network behaved coordinately when the nucleus was displaced. In elongated cells, where it was possible to deform the cell and the underlying cytoskeleton without deforming the nucleus, we observed that the actin cytoskeleton of the peripheral cytoplasm was also highly elastic. Fig. 2 shows an example in which an elongated REF cell transfected with GFP–γ-actin was subjected to a substantial deformation in the middle of an actin bundle along a concave webbed edge, which has been postulated to support part of the tension of cell adhesion . The induced deformations quickly recovered after release of the needle. This recovery was particularly impressive in that movement of the needle caused a small nick in the actin at the cell edge before the manipulation. It can be seen that this cut severed some actin filaments, in turn opening a gap in the margin of the cell. This indicates the margin is under tension, as expected. Further, the major deformation also clearly damaged the cell, causing parting of the cell immediately after elastic recovery. The damage sustained by the cell would be expected to dissipate part of the tension load, and thus act to suppress at least part of any elastic behavior. Complete elastic recovery by the actin cytoskeleton in the face of a dissipating influence was unexpected. We extended these observations of the elasticity and sustained tension on the actin network by manipulations specifically intended to sever the cytoskeleton. Microneedles were broken off 1–2 mm from the tip and the sharp, broken glass edge of such needles were then used as a microknife to cut into a cell, severing the cytoskeletal filaments in a local region of cytoplasm. Fig. 3 A shows a cell transfected with GFP–γ-actin subjected to a small nick at 0:07, again made in the middle of an actin bundle along a concave webbed edge. After this small cut, the needle was withdrawn completely. The images on the right side of the figure are difference images created by subtracting the pixels of the fluorescent image to the left with the next earlier image shown in the figure. As shown by both the fluorescent images to the left and the difference images to the right, the actin bundle retracts ∼12 μm on either side of the cut, indictating both tension and elasticity by the severed actin bundle. However, the difference images show that the effect of this release of tension was highly local: there is essentially no change in any other fluorescent actin bundle. Indeed, what appears to be a part of the severed actin bundle, extending toward the lower right corner of the cell, also shows little or no change in position for ∼1 min after. Fig. 3 B shows that the severed edge itself continued to remain stable for several minutes after the cut was made. However, 3–4 min after making the cut the entire right hand portion of the cell was observed to contract in a manner suggestive of increased tension on a catenary (e.g., pulling on the ropes of a simple rope suspension bridge). As shown by the difference image of Fig. 3 B, the cut edge of the cell increased slightly in diameter (i.e., the linear distance along the edge declined slightly) while the opposite, uncut cell edge moved in the same direction, e.g., as would the roadway suspended from the tightened rope suspension bridge. We presume that this cellular contraction was an active response to the cutting. Two GFP-tagged probes were used to visualize MTs. Transfection with GFP-tubulin works directly by incorporation of the fluorescent protein into the MT lattice. Microtubules were also visualized with GFP-MAP2c, a neuronal protein that binds to the outside of the lattice. Both probes have advantages and disadvantages. Despite the addition of the GFP component, GFP-tubulin does not appear to disturb MT structure or dynamics . It also seems unlikely that it would alter the MT array of the cell because cells have a translational-stage feedback mechanism that regulates the concentration of free tubulin subunits . Although this is advantageous for moderating the level of GFP-tubulin expression, it has the disadvantage that the labeled MTs are often too dimly fluorescent to visualize effectively. MAP2c is a well-characterized, high molecular weight, MT-associated protein of neurons that binds with high affinity to MTs . Expression and overexpression of this protein, labeled with GFP, in non-neural cells does not materially affect the dynamics of MTs , and results in a relatively large number of cells with brightly fluorescent MTs. Like MAP2c itself, however, GFP-MAP2c does cause bundling of MTs in those cells in which it is particularly highly expressed . We noted no differences in the mechanical behaviors of the cellular MT array between cells transfected with GFP-tubulin or GFP-MAP2c. In contrast to the elasticity of the actin cytoskeleton, the MT-based cytoskeleton when visualized with either probe recovered slowly from deformations and showed some degree of permanent deformation (i.e., deformations that persisted for the time scale of these observations) or flow. Fig. 4 is a REF cell transfected with GFP-MAP2c that was sharply poked in the nuclear region, which was surrounded by three bundles of MTs. The needle penetrated through and parted the cytoplasm such that the substratum was revealed, but without damaging the cell. After release of the needle, the MTs remained parted for >5 min, slowly filling the space made by the needle. Further, comparison of the position and curvature of the three perinuclear MT bundles before deformation by the needle (00: 09) with their geometry some 10 min after removal of the needle (14:33) show that these MTs have been displaced and remain displaced. Slow recovery after deformation and some degree of long-lasting displacement were typical of the response of GFP-illuminated MTs, whether via GFP-tubulin or GFP-MAP2. We assessed the response of the MT cytoskeleton to cutting using broken needles as before. Fig. 5 shows a fibroblast transfected with GFP-tubulin in which a deep cut was made in the cytoplasm, approximately perpendicular to the MT array, beginning in a particularly small radius concave region of the cell . Initially, MTs collected into a bundle at the tip of the needle . Subsequently, the cell fragment on the right was disrupted and lysed within 1 min accompanied by depolymerization of the MTs in the fragment. However, the cut edge of the cell to the left of the needle path retained its integrity with little change in the MT array in this region or in the extent of cell spreading at this cut edge. We confirmed the viscoelastic behavior of the MT cytoskeleton during substratum detachment and cell rounding of REF 52 cells transfected with GFP-tubulin. Cells that had been plated ∼16 h earlier were stimulated to detach and round up by adding EDTA to a final concentration of 0.1–0.5 mM. Two behaviors were routinely observed during subsequent retractions from the substratum; buckling of MTs in regions undergoing rapid retractions , and shearing flows in regions of slow retractions . Fig 6 shows a sequence of a rapidly retracting cell. As shown in Fig. 6 B, the cone-shaped extension toward the lower left of the cell retracted 20 μm in 2 min 39 s. (between 00:21 and 3:00) and is accompanied by obvious buckling of the MTs within this region. Importantly, the MTs begin buckling as soon as the margin of the cell began retracting. For example, between 00:21 and 1:10, the extension retracted only 5 μm, but there is clear buckling of the MT bundle near 11 o'clock. Fig. 6 C shows that the right side of this same cell undergoes an even more catastrophic buckling of MTs in response to the collapse of adhesion in this region, where the upper region of cytoplasm shown in Fig. 6 C retracts 15 μm in 11 s (between 02:49 and 03:00). In this and all other cases of MTs bearing new compressive loads, there was no evidence for MT disassembly stimulated by compressive forces, although we clearly observed MT assembly/disassembly in this cell characteristic of MT dynamic instability. Fig. 7 shows an example of a cell whose rounding up occurred more slowly. The lower region of the cell retracted 18 μm in 21 min accompanied primarily by shearing movements of MTs with only small amounts of buckling. Rounding up frequently occurs by a retraction of the MT-containing cytoplasm although the cell margin remains fully extended. Comparing the phase images in Fig. 7 , A and E, taken 41 min apart, indicates that the MT array initially extended to the outer margin of the cell, which did not change position, but the MT-containing cytoplasm has retracted some 20 μm apparently within the still extended cell cortex. Integrin-mediated cell attachment to a substratum also mediates mechanical attachments to the cytoplasmic actin cortex and has been shown to play a major role in regulating cytoplasmic architecture, cell shape, and motility . We examined the response of both the actin and the MT cytoskeleton to towing forces exerted by calibrated glass needles treated with laminin, an ECM protein known to bind to integrin molecules. Calibrated laminin-treated glass needles were applied under moderate force (∼200 μdynes) to the surface of REF cells transfected with GFP–γ-actin. In six separate experiments using different cells and different needles, we observed an accumulation of actin in the cortex beneath and surrounding the needle tip within minutes of applying the needle . If the needle was moved along the cell surface, applying forces <200 μdynes, the actin accumulation translocated with the needle. That is, moving the needle caused the accumulated actin to “walk” across the dorsal cell surface . The attachment of laminin-treated needles to the cell surface is mechanically robust. In the example shown in Fig. 8 , when the needle and actin reached the cell margin, a short extension could be pulled out and then rapidly yanked as shown in Fig. 9 . Difference images of these rapid and forceful deformations showed that only the actin within the short extension changed position , the observable actin throughout the rest of the cell did not change position or shape. Small experimentally induced extensions, as above, were observed to retract against even quite large tension loads imposed by a needle. In Fig. 10 , a fibroblast transfected with GFP-MAP2c that was in the process of cell spreading (replated ∼4 h before manipulation) was attached to the needle at the cell margin, which retreated and changed shape during the attachment process. After achieving attachment, the needle was pulled with gradually increasing force producing a short extension some 46 min after initial placement of the needle. By this time, the force in the extension was ∼1,000 μdynes. Against this load, the cell spontaneously retracted the extension from 00:46 to 1:01 (h:min), shown as the difference between positions marked 1 and 1′ in the figure. Between 1:01 and 1:24, the applied tension was increased to ∼3,000 μdynes and held. For the next 6 min, the cell again spontaneously retracted against this significant force load. By 1:31 of this same experiment, the reference needle required to assess tension in the needle attached to the cell was at the very edge of the optical field. This was the maximum load we could measure at this magnification. Accordingly, we switched to the lowest available objective (10×) in order to increase the size of the optical field and raised the force to near 7,000 μdynes (see below). As before, the cell spontaneously retracted induced lengthening of the cellular process (data not shown). At this point, some 20 min after the sequence shown in Fig. 10 , the reference needle had again been dragged to the edge of the optical field. To make an accurate measurement of force on the cell, we released adhesion of the cell to the calibrated needle by adding 60 μl of 0.5 mM EDTA in the vicinity of the needle tip allowing the needle to spring back to its zero-force distance. Based on the elastic recovery of a 16 μdyne/μm needle across 450 μm of the optical field, we estimate the cell and its process were supporting 7,200 μdynes or 7.2 × 10 −8 N. The diameter of this cell process varied between 2.3 and 3 μm at different times during this experiment. Accepting the largest diameter and a circular cross section gives an estimated stress of 10 5 dynes/cm 2 (= 0.1 bar = 1.5 psi), which is approximately an order of magnitude lower than the stress of a tetanically contracting skeletal muscle. The behaviors of the MTs during process extension and retraction was of some interest. First, a small number of MTs appear to be present in the experimentally induced cell process of Fig. 10 . More importantly, however, the images show very little change in the position or shape of the cell itself or of the nucleus. Further, there was little change in the prominent MT bundles seen within this cell. Particularly noteworthy is the bundle nearest the cone-shaped, mechanical anchor region of the experimentally induced extension. This bundle of MTs deformed very little throughout this sequence despite the substantial forces that are being exerted on a neighboring local region. Two types of control experiments were conducted for “towing” experiments using laminin-treated needles. Untreated needles (data not shown) were applied to the surface of the cell but never formed detectable attachments, nor was GFP-labeled actin observed to concentrate at the application site of such needles (or anywhere else). Needles were also treated with polylysine. These were found to stimulate recruitment of GFP–γ-actin to the site of the needle, albeit more slowly than for laminin-treated needles, and the connection was strong enough to permit “walking” of the actin accumulation across the cell surface . However, these polylysine-mediated attachments were relatively weak, detaching at applied tensions between 100–200 μdynes (1–2 × 10 −9 N). Two fundamentals for a mechanical understanding of any complex structure, e.g., a machine or a cell, are the mechanical behaviors of substructures composing the complex object and their interconnections. The development of GFP technology for cytoskeletal proteins enabled us to make some direct observations of the actin and MT cytoskeletons in response to applied mechanical forces. Our goal was not to examine the rheology of the cell in the usual sense of quantitative coefficients of fluid/solid stiffness, but to understand better the behaviors, e.g., the flow field, of the cytoskeleton and its interconnections in response to a variety of simple mechanical interventions. Indeed, these observations were most informative in assessing the time scale and spatial range over which the cytoskeleton changed or maintained form in response to forces that were of the same magnitude that these fibroblasts themselves exert. When a probe without attachment to the underlying cytoskeleton was used to apply force, we found that these attached cells behaved as predicted by the three-layer model of Dong et al. . The cell appears as a highly elastic nucleus that is surrounded by cytoplasmic MTs that behave like a viscoelastic fluid (e.g., jelly). The third and outermost layer is an elastic cortical actin shell with a sustained tension (pre-stress in the actin structures). The stiffness of this layer increased markedly when the experimental needle was treated with laminin to recruit the actin cytoskeleton to the surface. By directly visualizing the actin recruitment, we confirmed a widely postulated model for mechanical connections between integrins and the actin cytoskeleton. Whether the probe applied simple deformations to the cell or interacted with the cytoskeleton, we found little evidence for strong connections between the actin cortex and linear elements of the cytoskeleton, either stress fibers or the underlying MT network. That is, we observed that experimentally applied forces produced unexpectedly local responses by the cytoskeleton. In this regard, we found no evidence for a complementary force interaction between prestressed actin and compression-bearing MTs. In one experimental series , needles were used to poke, prod, and cut the cell while we observed the deformations of the actin and MT cytoskeleton. The nucleus was highly compliant and highly elastic; poking with a needle caused the nucleus to undergo substantial local deformations that recovered very rapidly after release of the needle . The contribution of the nucleus to the deformability of the cell has received very little attention, e.g., no mention in several recent reviews and monographs on cytomechanics . Our observations suggest that the properties of the nucleus are likely to play a significant role in the mechanical responses of cells, particularly the central region of attached cells. As previously shown by Maniotis et al. , we observed that the nucleus is stabilized in position by the actin cytoskeleton. Our observations did not allow us to determine whether this was by attachment or by steric entanglement, but movement of the nucleus clearly caused equivalent movements in the surrounding actin network, which also behaved elasticially . Indeed, our results for the mechanical behavior of actin are entirely consistent with the well-established view of the cortex as being an elastic structure under sustained tension or prestress . Actin observed in our transfected REF cells recovered its shape and position after noninjurious deformation over the course of seconds indicating nearly pure elasticity . Sustained tension was clearly indicated by the behavior of actin to cutting, in which the severed actin bundle retracted strongly with most change again occurring over the course of seconds followed by only minor changes over the course of the following minutes. The sustained tension in the cortex is presumably balanced by positive fluid pressure in the cytoplasm, which also provides resistance to poking, but this appears not to be a large force in relation to cytoplasmic viscosity insofar as no cytoplasmic spillage followed any cutting intervention. However, the high degree of localization of the actin response to pushing, prodding, and cutting was surprising given the widespread view of an integrated cytoskeletal network . By and large, only the actin filaments in the very immediate region of the intervention showed a response. These highly local responses to major changes in the form and/or connections of the cytoskeleton are inconsistent with complementary force interactions between tensile actin and compressed MTs, which would be predicted to promote more widespread rearrangements . In contrast to the elastic behavior of the nucleus and actin network, MTs behaved as a viscoelastic fluid and we observed little evidence for tethering among MTs or between MTs and the overlying cortex. That is, rapid deformations produced elastic, solid behaviors while deformations on a longer time scale produced flow and permanent deformations. The most dramatic solid-like behavior of MTs occured when they buckled in response to rapid retractions of cellular regions where the MTs were arrayed axially to the direction of retraction but even this buckling could result from cytoplasmic flow around floating MTs (e.g., like noodles in stirred soup). In all instances of buckling, no evidence for compression-induced MT disassembly was noted, although continued dynamic instability was observed. This suggests that either there was no compressive force on MTs, or that force does not directly regulate assembly/disassembly of MTs , at least in fibroblasts. Because the buckling of MTs began almost immediately on cytoplasmic retraction, i.e., when the compressive force would seem to be not much greater than before retraction, we suggest that the ability of fibroblast MTs to bear a compressive load is quite weak. Nor did we ever observe a concerted or organized shift in the MT array suggestive of an integrated arrangement of MTs that distributed an increased compressive load throughout the array. Instead there was only random buckling and on even a slightly longer time scale (min), MTs showed clear fluid behaviors. In the relatively slow cellular retraction of Fig. 7 , MTs primarily flowed past one another rather than buckled to accomodate the cytoplasmic movement. Even the MAP-induced bundle of MTs of Fig. 4 , where bundling would be expected to increase any elastic stiffness, showed only partial recovery of deformation. When MT bundles were manipulated directly by the needle, the bundles moved differently indicating a lack of interconnection, and were deformed in shape and position over the time scale of 10 min, dramatically different from the elastic recovery within seconds for actin responses. In rounding cells at the beginning of the formation of retraction fibers , the MT cytoskeleton retracted independently of the overlying cortex, which remained attached at the same sites on the substratum indicating a lack of interconnection between the MTs and actin cortex. When large numbers of MTs were severed in a spread cell , there was little or no long-range response. The cytoplasm at the cut edge of the living fragment behaved as if one had used a knife to cut through agar. We note that the rapid lysis of the severed fragment shown in Fig. 5 indicates that, except in this case, our manipulations did not significantly damage the experimental cells. Thus the responses of the cytoskeleton we observed cannot be ascribed to necrotic events. Indeed, cells are known to survive mechanical insult rather well, due in part to the capacity of the plasma membrane to reseal rapidly . Our results suggest that the localized mechanical responses of the cytoskeleton may also make an important contribution to injury resistance, e.g., the lack of wound spreading in Fig. 3 despite the cortical tension. This simple three-layer behavior for the “passive” rheology of the cell became more complex and active when the needle was treated with laminin and used to recruit actin to the cell surface, presumably through integrins . Actin remained elastic in these experiments, but seemed more like a rigid, solid gel than like the relatively compliant cellular structure seen with untreated needles. It is clear from Figs. 8 – 10 that laminin-treated needles form strong attachments to the cell surface and the actin array, but despite the application of substantial forces, the deformation of the cell and cytoskeleton were both very local. In Fig. 10 , the cell retained its locally deformed shape in the face of maintained and actively generated forces for a time scale of 100 min, as expected for rigid solids. We observed no integrated, wide-spread changes in the position of cytoskeletal or other cellular components. In the examples shown in Figs. 9 and 10 , pulling on the cell margin caused only a local change in cell shape, the formation of a cellular process. Neither the actin nor the MTs in regions neighboring the extension were altered by changes in the length or position of the extension itself nor did the cytoskeletal filaments in these regions show significant responses to changes in the forces exerted nearby. Again, these behaviors and their time scale suggests the sort of viscoleastic behavior typical of rigid solids, i.e., pulling produces only local necking without long range structural rearrangement. We presume that the contractions we observed by the experimentally induced cell processes are actomyosin-based contractions of the cortex, roughly similar to the well-described contraction of fibroblasts in collagen gels and on deformable growth substrata . The largest force we measured in this example was consistent with a recent measurement of the force generated by fibroblasts during locomotion and with contractile force exerted by essentially spherical fibroblasts . The stress across the induced cellular process was similar to that of fibroblasts strongly stimulated to contract with thrombin . In sharp contrast to cultured neurons that show a fluid-like growth response to tension and contract when slackened , fibroblasts responded to experimental extension with contractions of increasing force. We think it likely that by experimentally applying forces with laminin-treated needles, we engaged adhesion, deformation-sensing, and tensile-response machinery normally engaged in the wound-closure function of fibroblasts . We found the events of needle attachment interesting of themselves, although we can provide only a preliminary and incomplete interpretation. First, we observed a local accumulation of actin in the cytoplasmic region corresponding directly with the extracellular site of the laminin, an important ligand for integrins. This lends support to a widely accepted model of integrin-mediated attachment: that ligand binding to integrins causes a mechanical connection to the underlying cytoplasmic actin network . We were nevertheless surprised by the rapidity with which a visually dramatic accumulation of actin occurred and the strength of the connection between laminin and the cell surface. The adhesion between the cell and the tip of the needle shown in Fig. 10 was demonstrated to bear forces on the order of 10 −8 N for >1 h. There is no reason to assume that this represesents the upper limit of adhesive force, rather it was the largest force we could measure under the experimental circumstances. Intriguingly, the cortical actin accumulation at the needle could be dragged across the cell with only modest forces, again suggesting a lack of strong connections between the actin cortex and the underlying cytoplasm. The ability to pull out cellular extensions with larger forces indicates that under some conditions the connections between the extracellular adhesion protein (i.e., laminin) and the actin cortex are quite strong, e.g., able to resist stresses ∼1/10 that of contracting muscle. In this regard, Choquet et al. have shown that tension increases the strength of cytoskeletal connection to integrin receptors at lower forces (∼10 −11 N) and it may be that similar stress hardening occurred during our interventions. However, these tentative conclusions will require further study as the mechanical aspects of cellular attachment are currently less well understood than the underlying chemistry. In control experiments for laminin-treated needles, we found that polylysine-treated needles, but not untreated needles, also caused an accumulation of actin that was capable of being translocated beneath the cell surface. However, the attachment was not nearly as strong as with laminin. Cytoskeletal involvement and the architecture of polylysine-mediated adhesion has not received much attention, presumably because polylysine is a nonphysiological, nonspecific adhesion protein. Our results suggest that polylysine also causes actin assembly beneath the surface site of adhesion. With our method of applying tension, however, polylysine-mediated adhesion was considerably weaker, we would estimate by an order of magnitude, than laminin-integrin adhesion. Our results are difficult to reconcile with a tensegrity model of the cell in which sustained tension in the actin network is supported in part by compression of underlying MTs . Nor do our data provide any support for the related but separate idea that such a complementary force interaction directly regulates MT assembly/disassembly dynamics, at least in fibroblasts . As implied by the origin of its name, tensegrity structures are those in which the tensional elements behave in an integral fashion to provide the large-scale shape of the structure; i.e., tension creates large-scale integrity . Classic tensegrity structures also involve intimate and distributed connections between the overlying tensile network and internal compressive struts so that changes in the network produce changes in the array of struts. Yet we repeatedly observed that both the actin- and MT-based cytoskeleton responded only locally to either passive deformations or those in which the needle was attached to the actin cytoskeleton. In view of the direct evidence that some of the actin-based tension is borne by attachments to the dish (e.g., cells round up when detached from it), if MTs were compression-bearing elements in fibroblasts, then relatively rounded cells would tend to have more compressive force supported by MTs and the cortex would be more likely to bear on the MTs. For this reason, we manipulated both well-spread cells and cells that had been replated 2–6 h earlier and so were relatively rounded and in the process of forming strong attachments . We observed no differences in the cytoskeletal behaviors of rounded cells or well-spread cells in response to manipulation. Regardless of apparent degree of spreading and presumably attachment, the cytoskeletal responses to deformation were surprisingly local. In this regard, Thoumine and Ott conducted rheological measurements on essentially spherical chick embryo fibroblasts. Although no cytoskeletal inferences could be drawn, the overall behavior of their highly rounded cells were entirely similar to those reported here for fibroblasts of varying degrees of spreading: highly elastic responses over the time scale of seconds, viscoelastic responses over 5–15 min, and an active contractile response with adhesive conditions. In aggregate, these results indicate that fibroblasts do not change their qualitative, and possibly quantitative, mechanical properties depending on their shape or degree of spreading, as would be predicted of a tensegrity structure. The use of GFP-technology to visualize the cytoskeleton of living cells in real time adds an additional dimension to cellular rheology and cytomechanics. This technology makes it possible to directly observe the behaviors and interconnections of cytoskeletal elements in response to changes in cell shape and activity. We hope to exploit this technology in the future to better understand the cytoskeletal mechanics underlying cell crawling and the slower changes of cell shape change. | Study | biomedical | en | 0.999998 |
10189373 | All reagents were of analytical grade or higher and were purchased from Sigma Chemical Co. unless otherwise noted. FITC-Tfn was purchased from Molecular Probes Inc. 125 I was purchased from Amersham . Human holo-Tfn was purchased from Sigma Chemical Co. Human dIgA was a kind gift from J.P. Vaerman and Yves Sibille (Brussels, Belgium). Tfn and dIgA were radioiodinated using Iodogen to specific activities of ∼5 μCi/μg. MDCK cells transfected with human Tfn receptor (in pCB6 maintained with G418 selection) and rabbit pIgR (in pCEP4 maintained with hygromycin selection) were grown in DMEM plus 10% FCS. Cells were split 1:10 every 5–7 d. Confluent cells from a T-75 culture flask were trypsinized and plated into 2 × 75 mm Transwell cell culture inserts (Costar/ Corning). After 3 d, the cells were induced with 10 mM butyrate for 16 h. Cells were then kept in serum-free media at 37°C for 30 min before transfer into ice cold PBS 2+ (PBS with 0.9 mM CaCl 2 , 0.5 mM MgCl 2 ). Inserts were removed and placed onto 250 μl of 10 μg/ml labeled Tfn or dIgA in 1.5% (wt/vol) BSA for 1 h on ice. Cells were then washed five times for 5 min each in ice-cold PBS 2+ . Transwells were then placed in empty culture dishes in a 37°C water bath. Warm DMEM containing 100 μg/ml unlabeled holo-Tfn was added to the upper and lower chambers. Transwell units were removed to ice-cold PBS 2+ after 2.5, 20, or 25 min. Surface bound Tfn was removed with two successive 5-min washes in acid citrate (25 mM citric acid, 24.5 mM Na citrate, 280 mM sucrose, pH 4.6) and PBS 2+ . For dIgA, surface bound ligand was stripped with washes of DMEM plus 10 mM phosphate brought to pH 2.95. Cells were then scraped with a rubber policeman into 1 ml ICT (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 10 mM EGTA, 50 mM Hepes/KOH, pH 7.0) plus 250 mM sucrose, and passed four times through a ball bearing homogenizer using a 0.2496 inch ball bearing. A PNS was generated by centrifugation at 1,000 g for 5 min. The PNS was placed on a 5–20% (10–20% for immunoblots) linear Optiprep (Nycomed) gradient formed in ICT using a Gradient Master (Biocomp). Gradients were spun in an SW41 ultracentrifuge head (Beckman) for 18–20 h at 27,000 rpm (100,000 g at the tube bottom) at 4°C. Gradients were harvested using an auto Densi-Flow gradient harvester (Buchler). Gradient linearity was checked by refractive index. The plasma membrane was localized by alkaline phosphodiesterase activity . For single cohort internalization studies, MDCK cells transfected as above were sown at confluence on clear Transwell filters (0.4 μm pore) and incubated 3 d to polarize. Cells were induced with 10 mM butyrate overnight. Cells were incubated in serum-free media for 45 min to deplete Tfn. FITC-Tfn and/or Texas red–dIgA (produced from dIgA above using Texas red labeling kit from Molecular Probes) was bound to the basolateral surface at 0°C for 1 h. The cells were washed for 5 min, five times with PBS supplemented with ice-cold PBS 2+ as above. Ligands were internalized for either 2.5 or 25 min in DMEM media containing 100 μg/ml unlabeled Tfn and 10 mM Hepes. The cells were fixed in 3% PFA in PBS and then mounted in Moviol ( Calbiochem ) containing DABCO ( Sigma Chemical Co. ). Images were acquired with an excitation wavelength of 495 nm and an emission wavelength of 520 nm with either a 100× or 63× objective using a Axioplan microscope ( Carl Zeiss ) equipped with a digital camera ( Princeton Instruments ). The microscope and camera were driven by Openlab 1.7.6 (Improvision) run on an Apple Macintosh 9600. For preloaded cells and chase studies, MDCK cells transfected as above or CHO cells transfected with the human TfnR (in pCB6 maintained with G418 selection) were grown on 1-oz coverslips in DMEM (MDCK) or aMEM (CHO). Cells were sown thinly and induced while subconfluent using butyrate for 12–16 h. Cells were preincubated in serum-free media for 30 min at 37°C to deplete Tfn. Coverslips were then inverted onto droplets of 100 μg/ml FITC Tfn in serum-free media and incubated at 37°C for 30 min. Label was chased out of the cells with 100 μg/ml unlabeled holo-Tfn in the presence or absence of AlF 4 (50 μM AlCl 2 , 30 mM NaF) for up to 30 min. Cells were fixed in 3% PFA in PBS and then mounted in Moviol ( Calbiochem ) containing DABCO ( Sigma Chemical Co. ). Images were acquired with an excitation wavelength of 495 nm and an emission wavelength of 520 nm using an Axiophot microscope with a 63× objective and 100 ASA Ectachrome film ( Kodak ). Confocal images were acquired on a Bio-Rad 1050 confocal microscope with an excitation wavelength of 495 nm and an emission wavelength of 520 nm and digitally processed. Images were enhanced and combined using Adobe Photoshop on a Macintosh computer. Fractions from Optiprep density gradients were mixed with an equal volume of H 2 O containing 2% (wt/vol) sodium deoxycholate ( Sigma Chemical Co. ) and vortexed. Each fraction was brought to 10% trichloroacetic acid (Brand Nu Laboratories). After 30 min on ice, the precipitate was collected by centrifugation and the pellets washed for 30 min in two changes of 80% acetone (vol/vol in H 2 O). The resulting pellets were dissolved in SDS sample buffer and analyzed by SDS-PAGE followed by Western blot using ECL ( Pierce Chemical Co. ) detection. Cells from approximately half of a 75-mm Transwell were used for each set of blots. Rab4, cellubrevin, and TfnR antibodies have been described previously and Rab11 antibody was purchased from Zymed. Fractions from Optiprep gradients were directly fixed in Optiprep with 3% paraformaldehyde overnight at 4°C. Vesicles were adhered to grids by floating carbon-coated formvar-nickel EM grids on fixed fractions for 10 min. The grids were then blocked with 1% (wt/vol) fish skin gelatin and washed in PBS. 0.4% uranyl acetate/1.8% methyl cellulose ( Sigma Chemical Co. ) was then applied for contrast. First primary antibodies (same as for Western blots) were applied at 1:10 dilution in PBS/1% BSA, and a bridging antibody used for the polyclonal anti-rab4 antibody. 5-nm gold labeled secondary antibody was then applied (H. Geuze). The grids were washed and further fixed with glutaraldehyde. The process was then repeated with a second set of primary antibodies and 10-nm gold (H. Geuze). Colocalization was evaluated on a Phillips transmission electron microscope by counting at least 100 gold-labeled membrane objects (as well as unlabeled objects) in each specimen. MDCK cells were grown and seeded onto Transwell inserts as for density gradients except that 24-mm Transwell inserts were used with one-tenth the number of cells used for a 75-mm insert. Cells were induced and label bound to the basolateral surface at 0°C as for density gradients. After binding and washing, cells were warmed by addition of 37°C DMEM plus 100 μg/ml unlabeled Tfn to the upper chamber. After 1.5 min, this media was added to the lower chamber as well. The media from the upper and lower chambers was removed and replaced every 2 or 5 min for up to 90 min. Released 125 I-Tfn or dIgA was detected using a gamma counter (Beckman). In cases where the surface bound ligand was monitored, separate Transwell inserts were used for each time point. At the selected time, the Transwell was rapidly removed to ice-cold PBS 2+ and acid washed as for density gradients. The acid and neutral washes were combined for counting. Transwell membranes were cut from the inserts and counted directly to determine the amount of internalized label either at a selected time point or at the end of a series of time points. Because Tfn and dIgA remain associated with their receptors throughout a single round of endocytosis, the presence of ligand could be used to follow the progress of receptors through the cycle. The association is essentially quantitative so that dissociation constants are not included in the model. Both Tfn and dIgA are released upon reaching the surface. In the case of Tfn, reuptake was prevented by using excess unlabeled Tfn. Each data set was used individually for curve fitting and modeling. Kinetic values derived from each data set were then averaged to obtain the final rate constants. The models were based on the assumption that ligand moving from compartment A to compartment B does so in a first order manner with rate constant k (in units min −1 ) so that: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\begin{matrix}{\mathit{k}} \enskip & \\ {\mathit{A}}{\rightarrow}{\mathit{B}} \enskip & \end{matrix}\end{equation*}\end{document} is represented as dA / dt = kA ; conversely, dB / dt = − kB . Integrating this gives ln ( A t / A o ) = − kt , which converts to A t = A o e − kt . This final value represents the change in concentration of ligand in A over time t . Therefore, for ligand moving from A to B, the concentration of ligand in each after a unit of time is represented as dA / dt = − kA , integrated to give A t = A o e − kt ; dB / dt = kA , integrated to give B t = B o + ( A o − A o e − kt ). This equation can be carried on to multiple linked compartments A − k 1 → B − k 2 → C where after time t , the concentration of ligand in each compartment can be represented in an already integrated form as: A t = A o e − k 1 t ; B t = B o e − k 2 t + ( A o − A o e − k 1 t ); and C t = C o + ( B o − B o e − k 2 t ). The implicit assumption in these equations is that material that departs A for B during a time interval will not be immediately available for reexport from B to C (or back to A for that matter) during the same time interval. This can be viewed physiologically as the time in which ligand is sorted into a transport vesicle but has not yet arrived at the destination compartment. This time interval was arbitrarily set to 0.16666 min which is rapid in comparison to recycling kinetics. The complete set of equations was then recalculated after each time increment. For the model of recycling and transcytosis, the variables were defined as: A, material bound to basolateral membrane; a, a specific subset of bound dIgA that does not internalize; B, first endosomal compartment; C, second endosomal compartment; D, basolaterally recycled ligand; E, apically released (transcytosed) ligand; k 1 , rate of transport of ligand from A to B; k −1 , rate of transport of ligand from B to A; k 2 , rate of transport of ligand from B to C; k −2 , rate of transport of ligand from C to B; k 3 , rate of transport of ligand from C to D; k 4 , rate of transport of ligand from B to D; k 5 , rate of transport of ligand from C to E; k 6 , rate of transport of ligand from B to E; k 7 , rate of transport of ligand from a to D. The assignment of rate constants is graphically displayed in Fig. 6 . The equations describing transit of ligand were integrated as described above to yield the following algebraic forms for calculation: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{A}}_{{\mathit{t}}}={\mathit{A}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}1{\mathit{t}}}+{\mathit{B}}_{{\mathit{o}}}{\mathit{e}}^{-({\mathit{k}}-1){\mathit{t}}}\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{a}}_{{\mathit{t}}}={\mathit{a}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}7{\mathit{t}}}\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{B}}_{{\mathit{t}}}={\mathit{B}}_{{\mathit{o}}}({\mathit{e}}^{-[({\mathit{k}}-1)+{\mathit{k}}2+{\mathit{k}}4+{\mathit{k}}6]{\mathit{t}}})+({\mathit{A}}_{{\mathit{o}}}-{\mathit{A}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}1{\mathit{t}}})+({\mathit{C}}_{{\mathit{o}}}-{\mathit{C}}_{{\mathit{o}}}{\mathit{e}}^{-({\mathit{k}}-2){\mathit{t}}})\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{C}}_{{\mathit{t}}}={\mathit{C}}_{{\mathit{o}}}({\mathit{e}}^{-[({\mathit{k}}-2)+{\mathit{k}}3+{\mathit{k}}5]{\mathit{t}}})+({\mathit{B}}_{{\mathit{o}}}-{\mathit{B}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}2{\mathit{t}}})\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{D}}_{{\mathit{t}}}={\mathit{D}}_{{\mathit{o}}}+({\mathit{B}}_{{\mathit{o}}}-{\mathit{B}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}4{\mathit{t}}})+({\mathit{C}}_{{\mathit{o}}}-{\mathit{C}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}3{\mathit{t}}})+({\mathit{a}}_{{\mathit{o}}}-{\mathit{a}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}7{\mathit{t}}})\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{E}}_{{\mathit{t}}}={\mathit{E}}_{{\mathit{o}}}+({\mathit{B}}_{{\mathit{o}}}-{\mathit{B}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}6{\mathit{t}}})+({\mathit{C}}_{{\mathit{o}}}-{\mathit{C}}_{{\mathit{o}}}{\mathit{e}}^{-{\mathit{k}}5{\mathit{t}}}).\end{equation*}\end{document} These equations were entered into a spreadsheet program (Microsoft Excel 5.0) and used to model recycling and transcytosis. The initial values were set so that all of the internalized ligand started in compartment A (or a). In situations where some ligand was nonspecifically bound to the Transwell support (e.g., 5%) the starting value of compartment A could be set to 95%. Curves were manipulated by changing the values for the rate constants to fit each data set. The spreadsheet had a cumulative rounding error of <1.5%. To model a pathway that does not exist or is completely inhibited by a drug, the k for that pathway was set to zero. As may be expected, the early, middle, and late portions of curves representing transcytosis and recycling were differentially sensitive to changes in different rate constants, allowing derivation of unique solutions to each curve fitting situation. We and others have demonstrated the existence of distinct peripheral EE and perinuclear RE populations that label after the uptake of Tfn in nonpolarized CHO cells . To confirm that analogous endosome populations exist in polarized epithelial cells, we monitored the endocytosis of a synchronous pulse of FITC-Tfn by filter-grown MDCK cells transfected with the human TfnR. After FITC-Tfn was bound at 0°C, the cells were washed and then warmed to 37°C for 2.5–25 min to permit endocytosis. After fixation, deconvoluted optical sections were taken in the basal versus apical cytoplasm (0.5 vs. 4.2–4.5 μm from the filter surface, respectively). As shown in Fig. 1 , after 2.5 min of uptake much of the FITC-Tfn was in small vesicles scattered throughout the basal cytoplasm (B) and, to a lesser extent, in the apical cytoplasm (A). After 25 min, less total FITC-Tfn remained and its distribution was markedly different. Most labeled structures were now found in the apical cytoplasm, clustered around the nucleus . As compared with the pattern at 2.5 min, the basal cytoplasm was relatively devoid of labeling . We operationally defined the vesicles labeled after 2.5 min as EEs and the apical structures labeled after the 25-min chase as REs. Identical results were obtained if the cells were pulsed and chased entirely at 37°C indicating that the differential localization of Tfn-containing endosomes was not induced by the temperature shift (not shown). Although Tfn clearly labeled geographically distinct populations of vesicles at different times of uptake, this criterion alone cannot reliably distinguish between EE and RE populations. Even when supplemented by the immunofluorescence data showing a differential distribution of rab proteins between the two populations, the distinctiveness of compartments must be supplemented by physical separation. Thus, to determine if EEs and REs represent distinct compartments, we sought to separate them by cell fractionation. 125 I-Tfn was bound to the basolateral surface of filter-grown MDCK cells at 0°C and then internalized at 37°C in the presence of unlabeled Tfn for various times. At each time point, the cells were washed in low pH buffer (to remove surface bound 125 I-Tfn), homogenized, and postnuclear supernatants centrifuged for 20 h in preformed 5–20% Optiprep gradients. When cells were fractionated after 2.5 min of uptake, a major peak of 125 I-Tfn was observed at ∼15% Optiprep (fractions 24–26). A minor lower density peak (fractions 6–9) was also observed. Since the low density peak comigrated with phosphodiesterase activity and with 125 I-Tfn from cell homogenized without warming, it was likely to represent remaining plasma membrane-bound ligand. Thus, the major peak (fractions 24–26) must represent the EE population. When 125 I-Tfn was chased into the cells for 25 min before homogenization, centrifugation yielded a single peak that was shifted to a slightly lower density than the 2.5 min EE peak . Since this second peak appeared at the expense of the EE peak and only after >20 min of chase, it was likely to correspond to the apical RE population in MDCK cells. Although the kinetically defined EE and RE peaks were not completely separated under the gradient conditions used, they were clearly resolved, demonstrating that EEs and REs are physically distinct entities. We next characterized the EE and RE peaks using markers which might be differentially distributed between the two endosome populations. For this purpose, we centrifuged MDCK cell postnuclear supernatants on shallower 10–20% Optiprep gradients after labeling with 125 I-Tfn for 2.5 or 25 min. In addition, surface bound Tfn was not removed before homogenization in order to provide a convenient plasma membrane marker. Although both peaks were somewhat broader, EEs (2.5 min) were clearly detected at fraction 23 while REs (25 min) were at fractions 19–20 . The equilibrium distribution of the TfnR was probed by Western blot. As shown in Fig. 3 B (top row), most of the TfnR sedimented with REs (fractions 19–20) with a well resolved small peak in the low density region of the gradient (fractions 2–7). This low density peak was likely to reflect plasma membrane TfnR, as it cosedimented with membranes labeled when 125 I-Tfn was bound at the basolateral surface before homogenization. Thus, the majority of the TfnR detected on the Optiprep gradients was associated with intracellular membranes, especially REs. We next determined the distribution of rab4 and rab11, two rab proteins which have been associated with EEs and perinuclear REs by immunofluorescence. The presumptive EE marker rab4 was more closely associated with membranes labeled after the 2.5-min pulse of 125 I-Tfn, exhibiting a peak at approximately fraction 23 and clearly resolved from the major TfnR peak . rab11, on the other hand, did not cosediment with rab4-containing EEs and was instead shifted to lower density membranes overlapping with the major TfnR peak (fractions 19–20). Although large fractions of rab4 and rab11 were detected in very low density regions (fractions 1–9), both rab proteins were judged to be soluble as they did not pellet after ultracentrifugation. In contrast, rab4 and rab11 in the endosome-containing fractions were quantitatively pelleted when centrifuged under identical conditions (not shown). We could not determine if the low density rab4 and rab11 reflected a large cytosolic pool in MDCK cells or if they were released upon homogenization (as are other peripheral proteins such as COPI and Arf). In either case, it is important to note that all of the membrane-associated rab4 and rab11 detected in the gradients cosedimented with the organelles with which they had been previously associated by immunocytochemistry. To demonstrate that TfnR and rab4 (or rab11) were present in the same vesicles, membranes were recovered from Optiprep gradient fractions, immobilized on EM grids, and labeled with antibodies to rab4, rab11, or the cytoplasmic domain of TfnR . Of the vesicles found that labeled with TfnR in fraction 24 of the EE peak, 63% were also positive for rab4 (TfnR labeled with 5-nm gold, small arrowheads; rab4 labeled with 10-nm gold, large arrowheads). 42% colabeled for TfnR and rab11 (10-nm gold, not shown). Approximately 40% of the vesicles in this fraction were positive for rab4 but negative for TfnR, presumably representing EEs depleted of TfnR. In the RE peak (fraction 27), 73% of the structures were double-positive for both TfnR and rab11, although 60% of the TfnR-positive vesicles were also positive for rab4. Only 16% of the structures in the RE peak labeled for rab4 alone, however. Thus, the overlapping but distinct Tfn-containing EE and RE peaks comigrated differentially with rab4 and rab11, demonstrating that the two endosome populations are physically and biochemically distinct. As a result of the homogenization and fractionation procedures, nearly all of the vesicles recovered from the gradients exhibited a nondescript round morphology , preventing any further conclusions based upon either morphology or localization of the immunolabel within each vesicle. Consequently, it was of interest to determine if EEs and REs had distinct functional attributes. To help clarify the relative functions of the two endosome populations, we sought to identify pharmacologic agents which selectively altered the transit of Tfn through either EEs or REs. Various potential inhibitors were screened for their ability to alter FITC-Tfn transport. We first used CHO cells since they organize their REs in a characteristic position directly over the nucleus . One useful agent was the phosphate transition state analogue AlF 4 , a phosphatase inhibitor and agonist of various GTPases , which appeared to slow the exit of Tfn from REs. This effect was illustrated in Fig. 4 . When CHO cells (transfected with the human TfnR) were pulsed with FITC-Tfn for 30 min, various small peripheral and perinuclear structures were labeled . These greatly decreased in intensity after a 30-min chase in unlabeled Tfn . However, when the chase medium contained 30 μM AlF 4 , the intensity of perinuclear labeling was significantly greater . Similar results were obtained for MDCK cells , although AlF 4 treatment did not appear to cause as much intracellular “retention” of FITC-Tfn (see below), nor were the labeled endosomes as well organized in a perinuclear array as in CHO cells. However, as previously found for dIgA-containing REs, the FITC-Tfn–containing REs were concentrated in the apical cytoplasm as revealed by vertical (X-Z) confocal sections . Moreover, fractionation on Optiprep gradients demonstrated that 125 I-Tfn remaining in MDCK cells chased in the presence of AlF 4 was limited to fractions characteristic of REs . These fractions were also devoid of markers for the Golgi complex (e.g., mannosidase II, galactosyl transferase; not shown), indicating that AlF 4 did not cause Tfn transport into cisternal Golgi elements. Thus, in both CHO and MDCK cells, AlF 4 appears to cause a transient accumulation or retention of FITC-Tfn in REs. Quantitative measurements of Tfn recycling in AlF 4 -treated cells confirmed the suggestion that the intracellular retention of Tfn was more pronounced in CHO than in MDCK cells. Whereas recycling was inhibited by ∼30% after 2 h in CHO cells, in filter-grown MDCK cells the total amount of recycling into both the apical and basolateral media was inhibited by only ∼10% . However, there was a marked alteration in the polarity of recycling. Although ∼95% of the internalized 125 I-Tfn was released back into the basolateral medium by control cells, when 30 μM AlF 4 was added during the recycling period, substantial amounts of 125 I-Tfn were now released into the apical medium . This induction of basolateral to apical transcytosis involved up to 20% of the total internal 125 I-Tfn after 40 min of chase, as compared with <5% transcytosis in control cells. Since AlF 4 may act preferentially at the level of REs, REs may thus be an important site for polarized sorting. To address the relative contributions of EEs and REs in polarized receptor recycling, we performed detailed kinetic analysis of basolateral recycling and transcytosis using MDCK cells stably transfected with both human TfnR and rabbit pIgR . 125 I-Tfn was bound to the basolateral surface at 0°C, and after rinsing the filter-grown monolayers were shifted to 37°C in the presence of excess unlabeled Tfn to prevent reinternalization. At 1–5-min intervals, the appearance of 125 I-Tfn in the apical medium (transcytosed) or basolateral medium (recycled) was followed, as was the presence of acid-sensitive 125 I-Tfn still bound to receptors on the basolateral surface . For some experiments, 30 μM AlF 4 was included during the 37°C incubation. The data were analyzed using a series of conventional mathematical models describing the transport of Tfn as a series of first order rate constants connecting the apical and basolateral surfaces with various internal compartments (equations in Materials and Methods) . We first determined the rate of internalization of surface bound 125 I-Tfn by measuring the disappearance of 125 I-Tfn that was sensitive to removal by acid stripping, as well as the appearance of 125 I-Tfn in the media and acid-insensitive 125 I-Tfn associated with the cells. Based on computer analysis of the kinetic data obtained (see Materials and Methods), we derived a rate constant for internalization of 0.21 min −1 ± 0.0077, similar to other cell types . There was no dissociation of Tfn into the medium, nor was the internalization rate altered by AlF 4 (not shown). We also measured the polarized recycling of 125 I-Tfn by monitoring its appearance in the apical or basolateral media at 2-min intervals. These data were compared with a simple kinetic model in which the intracellular transit of Tfn followed the “single” pathway generally thought to be followed by recycling receptors: internalization and delivery to EEs, from EEs to REs, and from REs back to the plasma membrane (basolateral or apical). Reversible rate constants were assigned to each of these steps . In the case of the initial internalization step ( k 1 ), the reverse rate ( k −1 ) would account for the possibility that some internalized Tfn–TfnR complexes might be recycled without reaching an acidified compartment and thus would remain receptor-bound after returning to the surface. Transit of Tfn to LEs and lysosomes was assumed not to occur, as shown previously . By systematically varying the values for each forward and reverse rate constant (holding k 1 at its experimentally determined value), predicted curves for basolateral and apical recycling were obtained and compared with the actual data . Curves generated by these equations were fit to experimental data sets so as to minimize the sum of the squared differences (SS) between predicted and experimental values . For control cells , the best fit curves matched the recycling and transcytosis data reasonably well, although there were slight variations between data and prediction (SS = 336 where SS = 0 is a perfect fit). More striking were the deviations between predicted curves and actual data in cells treated with AlF 4 (closed symbols). In this case, the best curve yielded SS = 836. Regardless of the combination of values employed for each of the rate constants, predicted curves for basolateral recycling never corresponded to the actual kinetics of recycling. Placing additional endosome compartments in series along a single pathway together with EEs and REs only served to further degrade the degree of curve fitting for both control and AlF 4 -treated cells (not shown). Thus, particularly in AlF 4 -treated cells, the widely assumed pathway of sequential transport of Tfn from plasma membrane to EEs, to REs, and then back to the plasma membrane did not fit a kinetic representation of the data. Similar results were obtained for endocytosis and recycling in nonpolarized CHO cells (not shown). Since a single pathway involving obligatory transit from EEs to REs before recycling did not yield kinetics that optimally matched the data, we asked whether recycling might occur directly from EEs as well. To test this possibility, we introduced a single additional rate constant, k 4 , to describe a potential rapid recycling pathway from EE directly back to the plasma membrane . Again, by systematically varying each rate constant, it was possible to identify solution sets which more accurately reflected the recycling and transcytosis data in control cells (SS = 140) and even cells treated with AlF 4 (SS = 64) . The fit of the two-pathway model versus the one-pathway model was tested using Fischer's F-test for goodness of fit where F = ([ SS 1 − SS 2 ]/[ df 1 − df 2 ])/( SS 2 / df 2 ) . SS refers to the sum of the squares of the variance at each data point and df refers to the degrees of freedom (number of data points minus number of nonfixed pathway values). For the two-pathway model as compared with the one-pathway model in control data F = 48 and for the comparative fits of AlF 4 -treated cell data F = 424. For the 40 data points in each set, 5 independent kinetic parameters in the single model and 6 independent kinetic parameters in the two-pathway model P < 0.005 when F > 11.46 . Thus, the addition of the second pathway gave a significantly better fit to the data than did the single pathway model. Table I summarizes the predicted rate constants used to generate the optimized curves. The most remarkable feature was the finding that the unique best solution for the Tfn recycling and transcytosis data in AlF 4 -treated cells required setting the rate of RE to basolateral transport ( k 3 ) to zero. Any non-zero value for k 3 predicted much greater slopes for Tfn recycling after 30 min than were actually observed. Non-zero values for k 3 also produced curves containing lower levels of Tfn retained intracellularly or recycled apically than were obtained experimentally. With k 3 at zero, a rate constant for direct recycling from EE ( k 4 ) was calculated to be 0.097 min −1 ± 0.020 (Table I ). Thus, a considerable fraction of the basolateral recycling that occurs in AlF 4 -treated cells was predicted to come directly from EEs. The remaining rate constants were derived by placing further constraints on the construct, forcing it to account at each time point for the rate and extent of apical recycling (transcytosis), and the amounts of Tfn remaining intracellularly, basolaterally recycled, and remaining on the basolateral surface. Using these constraints, we derived a unique solution for the rate of Tfn transport between EEs and REs . Interestingly, these values are similar to those derived from video microscopic measurements of transfer between geographically defined REs and EEs in CHO cells . We considered a possible contribution of a pathway ( k 6 ) from EEs directly to the apical surface, particularly in AlF 4 -treated cells. However, setting non-zero values for k 6 produced curves that fit less well than the unique solution provided by setting k 6 to 0 and k 5 to 0.027 min −1 ± 0.0028. In untreated control cells, the best curve fits were obtained using non-zero values for k 3 . Systematic generation of progress curves produced a value of 0.057/min ± 0.014 for k 3 . Remarkably, this was the only major difference between control and treated cells. The derived value in control cells for k 4 , direct recycling of Tfn from EE to the basolateral medium, was 0.11 min −1 ± 0.020, only slightly higher than that derived in the presence of AlF 4 ( k 4 = 0.097 min −1 ) (Table I ). This suggested that AlF 4 partially inhibited recycling from EEs, but nearly as much as from REs. It was also of interest that the calculated rate constant for transport from REs to the apical plasma membrane ( k 5 ) in control cells was comparable to that obtained in AlF 4 -treated cells, despite the fact that AlF 4 greatly increased the apical transcytosis of Tfn. Thus, the ability of AlF 4 to enhance transcytosis appears to result more from the selective inhibition of Tfn transport from REs to the basolateral surface than from an enhancement of endosome to apical membrane transport. The kinetic model of Tfn recycling was derived from measurements of 125 I-Tfn that was released apically, released basolaterally, remained bound to the basolateral surface, or was otherwise cell associated. Due to inaccuracies inherent in determining amounts of 125 I-Tfn in overlapping gradient fractions, we did not attempt to determine quantitatively the amounts of Tfn in EE and RE by cell fractionation at each time point. However, the model simulates passage through EEs and REs and thus makes specific qualitative predictions about the Tfn passage through these compartments. As shown in Fig. 7 A, EEs were predicted by the model to fill maximally at 5 min, as compared with 12–13 min for REs. At 2.5 min, the model predicted that >90% of the Tfn remaining in the cell will be in the EEs. At 25 min, although much of the Tfn would have already left the cell, the model predicted that >66% of the remaining intracellular Tfn was in REs. These predictions were in accord with the fluorescence microscopy as well as the cell fractionation data. AlF 4 treatment provided an additional way to check these predictions empirically. According to the model, there should be little change in the levels of Tfn in EEs, but at 30 min there would be a doubling of the Tfn remaining in the REs . This prediction is borne out by the observation that AlF 4 treatment caused selective if transient retention of FITC-Tfn in the REs. Additionally, the mathematical model predicts that AlF 4 will not alter the passage through the EE nor the nature of the compartments, a prediction confirmed by the subcellular fractionation of AlF 4 -treated cells . Thus, specific predictions of the model concerning the intracellular localization of Tfn at different time points could be confirmed by the fluorescence microscopy and cell fractionation data, neither of which was used in formulating the model. A limitation of the analysis using Tfn recycling was that much of our ability to kinetically dissect two recycling pathways was based on alterations due to AlF 4 treatment. We next examined the model's ability to describe the behavior of pIgR-bound dIgA, a ligand which is normally (i.e., in the absence of AlF 4 ) subject to recycling as well as transcytosis . Since the MDCK cells used for the Tfn uptake experiments were doubly transfected with pIgR, we were able to use the same cells for both ligands. In MDCK cells transfected with only the pIgR, basolaterally applied dIgA passed through endosomes accessible to Tfn internalized to equilibrium . We first established that the pIgR in the double transfectants behaved as did MDCK cells expressing only the pIgR. To monitor transcytosis, polarized filter-grown monolayers were allowed to internalize 125 I-labeled human dIgA from the basolateral side for 10 min at 37°C, washed, and then recultured in the absence of 125 I-dIgA for an additional 60 min. In agreement with previous results, ∼50% of the internalized 125 I-dIgA appeared by transcytosis in the apical medium by this time point (not shown) . Next, FITC-Tfn and Texas red–dIgA (TR-dIgA) were bound to the basolateral surface on ice, and the two ligands then were internalized for 2.5 or 25 min. In cells expressing both receptors, at 2.5 min FITC-Tfn and TR-dIgA colocalized in basolateral EEs . After 25 min, both ligands were colocalized in more perinuclear, apical REs . Cell fractionation was used to confirm the fluorescence microscopy. 125 I-dIgA and 125 I-Tfn were bound to the basolateral surface of MDCK cells as above. After 2.5 or 25 min of uptake, cell homogenates were prepared and centrifuged on Optiprep gradients. 125 I-dIgA was transferred between vesicle populations that cosedimented with Tfn-containing EE and RE both after 2.5 min and 25 min of chase, again suggesting that Tfn and dIgA transit through the same endosomal populations. We next measured transcytosis of 125 I-dIgA prebound at 0°C. Under these conditions, only 25% of the initially bound 125 I-dIgA appeared in the apical medium since at least half of the bound 125 I-dIgA dissociates into the basolateral medium before endocytosis. Since this process would affect the kinetic analysis of recycling, we first examined the rates of 125 I-dIgA internalization and dissociation . The rate of internalization was determined by measuring the amount of 125 I-dIgA becoming resistant to acid stripping at 1-min intervals after warm up. The rate of dissociation was determined by measuring release of 125 I-dIgA into the basolateral medium. 45% ± 3.7% of the bound 125 I-dIgA was in the internalizable pool, while 51% ± 2.7% was in the pool of IgA which dissociated before internalization. It was possible that internalized and dissociated dIgA originated from a single pool of surface bound dIgA. However, the comparative fit of the two-pool model to a one-pool model using the dIgA internalization data with Fischer's F-test as above yields F = 1200 (for F > 3.89, the value of P < 0.005) clearly providing a significantly better fit for the two-pool model of dIgA binding. From these data, we could calculate an internalization rate constant ( k 1 ) of 0.202 ± 0.011 min −1 , and more rapid rate of 125 I-dIgA dissociation into the basolateral medium ( k 7 ) of 0.712 ± 0.13 min −1 . The rate of 125 I-dIgA internalization was indistinguishable from that for Tfn (Table I ). Having determined the rate at which IgA dissociated without internalization, it was now possible to dissect this effect from recycling of internalized IgA into the basolateral medium. After binding of 125 I-dIgA as above, the cultures were warmed and the amount of 125 I-dIgA in the medium determined every 30 s . Over the course of 8 min, a biphasic curve was apparent. During the first 2.5 min, 125 I-dIgA appeared in the basolateral medium at the same rate calculated for rapid dissociation from the receptor (designated as k 7 = 0.712 min −1 ). However, by 4 min, the rapid rate of 125 I-dIgA appearance had ceased but radioactivity continued to accumulate in the basolateral medium. Analysis of this slow phase of 125 I-dIgA release over the next 4 min yielded a single rate constant of 0.095 ± 0.027 min −1 . Assuming that recycling at times <8 min resulted from recycling out of EEs rather than the REs, we presume that this rate must describe k 4 , the rate of dIgA recycling from EEs. Interestingly, this experimentally obtained value was very similar to the calculated value for Tfn recycling (Table I ). Using the measured values for internalization, dissociation, basolateral recycling, and transcytosis, we subjected the dIgA data to the same kinetic analysis as performed for Tfn. Again, the data were best described by the two-compartment pathway . In other words, the model in which basolateral recycling or apical transcytosis can occur from either EEs or REs provided kinetic curves which fit the data extremely well (SS = 19.6) , far better than curves obtained assuming that recycling and transcytosis can only occur after transfer to REs from EEs (F = 82.9; for seven parameters in this dual pathway model and six in the single pathway model, when F > 9.1, the value of P < 0.005). The most significant result was that the derived rate constant for recycling from RE to the basolateral medium ( k 3 ) was zero, as calculated for Tfn recycling and transcytosis in AlF 4 -treated cells (Table I ). The model would predict that AlF 4 would have no effect on dIgA recycling or transcytosis; this was indeed the case when tested experimentally (not shown). A comparison of the rate constants obtained for dIgA in untreated cells and for Tfn in AlF 4 -treated cells yielded few other significant differences (Table I ). The predicted rate of anterograde and retrograde transport between EEs and REs was increased, possibly suggesting that dIgA and Tfn may be sorted differentially in both compartments. Moreover, the model predicted a low level of EE to apical plasma membrane transport ( k 6 ) which was not seen for Tfn with or without AlF 4 . Thus, a two-compartment model can be used to accurately describe the endocytosis, recycling, and transcytosis of dIgA. The data also suggest that the ability of AlF 4 to increase Tfn transcytosis may in effect phenocopy a physiological significant sorting mechanism which enhances dIgA transcytosis by blocking dIgA entry into the same basolateral pathway inhibited by AlF 4 . It has long been appreciated that receptor recycling in polarized and nonpolarized cells alike involves two spatially distinct populations of endosomes, referred to here as EEs and REs . However, why this should be the case has never been clear, nor have the relative roles of EEs and REs in the recycling pathway been firmly established. Recent immunofluorescence evidence has suggested that the two populations differ in their rab protein compositions. In addition, given that REs rarely contain fluid phase markers of endocytosis, there is good reason to believe that they represent structures that occur after EEs have sorted receptors destined for recycling from dissociated ligands destined for transport to LEs and lysosomes. Beyond these features, however, the relationship between EEs and REs has remained elusive. In this paper, we have presented three lines of evidence that REs and EEs represent distinct populations, which can be resolved physically (by centrifugation), biochemically (by protein composition), and pharmacologically (by differential sensitivity to AlF 4 ). By performing a kinetic analysis in polarized MDCK cells doubly transfected with recycling and transcytotic receptors, we have been also able to dissect functional differences between the two populations. This was possible since use of MDCK cells allowed us to directly measure two distinct recycling pathways, basolateral recycling versus transcytosis, and to monitor the effects of various manipulations on their kinetic properties. Based on the physical and functional distinctiveness of EEs and REs, it is now appropriate to refer to them as compartments, although such references have appeared previously without the minimal requirements for defining compartments having been met . EE and RE have distinct protein compositions as indicated by the observations that TfnR and rab11 are found preferentially with REs while rab4 is found preferentially with EEs, by both Western blot and immunocytochemistry on the isolated fractions. Given the obvious pathway relationship between EEs and REs, however, it is expected that the distributions of any given markers between the two compartments will be less than complete. Our results also illustrate the distinct functions of EEs and REs, evidenced by both the distribution of TfnR and kinetic analysis of the transit of Tfn. At equilibrium, most of the internal TfnR is associated with the RE rather than the EE, despite the fact that EE appear to account for the large majority of recycling back to the basolateral surface. Based on the model, it appears that EE return >65% of basolaterally recycling TfnR as well as a significant fraction of receptors targeted for transcytosis. EE-mediated recycling also appears to be rapid, being completed within 10 min of internalization. Thus, from first principles, it now seems likely that the rapid phase of Tfn recycling occurs directly from EEs, rather than via rapid transit through REs as presumed previously. On the other hand, our model predicts that EEs have at best a minor role in sorting receptors to the apical surface. This activity appears to be the purview of REs. Although the model predicts that pIgR may be transferred from EEs to REs somewhat more efficiently than TfnR, sorting in the REs of pIgR-bound dIgA from TfnR is virtually complete. The kinetic analysis revealed that few if any dIgA– pIgR complexes are likely to be transported directly from REs to the basolateral plasma membrane, although a fraction are returned in a retrograde fashion to EEs (from which basolateral recycling presumably could once again occur). In contrast, very little of the Tfn that reaches RE is transferred to the apical surface, most of it being sorted basolaterally. It is precisely this sorting event that appears to be blocked by AlF 4 . By this view, then, the recycling pathway consists of two endosomal compartments which function in tandem to ensure the polarized recycling of receptors internalized from the basolateral surface. Certainly, our conclusions are limited by the fact that many important points were derived from kinetic analysis. In enzymology, it is well known that kinetics can never be used to assign precise reaction mechanism but only can be consistent with predicted models. Nevertheless, as sometimes occurs in enzymology, kinetic analysis can be the best or only possible strategy. Despite our ability to resolve EE from RE on density gradients, the degree of separation was incomplete, making it difficult to precisely quantify the localization of Tfn or dIgA to one or the other endosome compartment, particularly at time points when both tracers would be variably present both in EE and RE. It is highly likely that precise measurements would be needed, since targeting on the endocytic pathway is characterized by iterative low efficiency sorting events as opposed to single sorting reactions that result in complete separations at each cycle . Thus, we would anticipate that the relative concentrations of Tfn versus dIgA in EE or RE would not vary markedly, making direct determinations difficult even after separation methods are improved. Mathematical methods have been used in the past to describe recycling of Tfn and other ligands in a variety of cell lines. Although there have been some suggestions that multiple routes for recycling from the endosomes to the basolateral membrane may exist , most of these models assumed obligatory passage of ligand through the REs or rationalized the slower component of recycling in terms of nonvectoral retrograde flow between the two compartments . Our analysis has as its foundation the biochemical separation of two distinct endosomal populations. Thus, our approach involved fitting a series of accurate measurements for internalization and recycling into the minimum number of compartments known to be involved, rather than using kinetic modeling to predict the existence of the compartments themselves. This is a crucial point. Our model was defined by a series of first order rate constants that describe only the transfer rate of ligand from one compartment to another, without making any assumptions concerning the nature of the compartments. Curve fitting was performed by systematically varying each of the rate constants that must interconnect EEs with REs, as well as both compartments with the plasma membrane. By constraining the progress curves by four experimental measurements in each case (rates of surface clearance, endocytosis, basolateral recycling, transcytosis), unique calculated solutions could be obtained for each data set. Best fits were stringently evaluated, first by eye, then by minimizing the sum squared errors (a common regression fitting technique). Comparative fits of different models were then performed using the well characterized statistical method of the Fischer F-test using a table of F values and degrees of freedom to provide the P value for relative significance of the fit improvement between models. Our interpretation that the majority of basolateral to apical transcytosis reflects a sorting event in RE is consistent with recent results showing that rab17 is enriched in perinuclear RE structures in mouse mammary epithelial cells and that disruption of rab17 function by the expression of a dominant negative rab17 cDNA inhibits transcytosis but not basolateral recycling . There are, a priori, many mathematical constructs which could be designed to model our recycling and transcytosis data. However, any model must reflect not only the kinetic data for recycling and transcytosis of Tfn and dIgA, but also for the passage through distinct endosomal populations. It is instructive to consider several such alternatives. For example, as discussed above, a model invoking a single pathway for Tfn passing obligatorily through the EEs and REs before recycling cannot be made to fit the experimental data and thus can be ruled out. To date, this has been the most widely presumed model for Tfn recycling, certainly in nonpolarized cells . Similarly, models invoking a single endosomal compartment can be ruled out because they do not accurately fit the data for AlF 4 -treated cells , nor do they match the data demonstrating that Tfn passes through distinct compartments . Similarly, a model invoking two compartments with entirely distinct functions in basolateral recycling and apical transcytosis can be evaluated and eliminated. In this case, there is not direct passage from the RE to the basolateral surface, and AlF 4 might affect the retrograde RE to EE transport or partially affect transport from EE to the basolateral surface. Such a model can be nicely made to fit the experimentally derived kinetic data . However, it makes the prediction that the EE will contain as much or more Tfn than the RE in untreated cells even at 20 min . This prediction is not consistent with either the fluorescence or density gradient fractionation data. While a minimum of two compartments and two recycling pathways must exist (based on kinetics and cell fractionation), it is theoretically possible to add compartments between the plasma membrane and the EE or RE and still fit the experimental data. For example a compartment can be added between the basolateral membrane and the EE and the experimental data will still fit well . Also, the predictions for Tfn transport through EEs and REs Tfn content will be unaltered . However, the rate of exit from the newly added compartment must be close to 1.2 min −1 , severalfold more rapid even than the initial internalization step and not consistent with values derived from morphologic analysis . Additional compartments can be added along the exit pathways from EEs and REs to the plasma membrane only so long as the rate of exit from the EE or RE is not altered and the rate of exit from the added compartment exceeds the rate of entry into that compartment. As with the addition of compartments between EEs, REs, or the plasma membrane, addition of a compartment between EEs and REs cannot be ruled out . This model can be adapted to fit the experimental kinetic Tfn recycling data in control cells (not shown) and in AlF 4 -treated cells . However, it predicts that addition of AlF 4 will result in an accumulation of one-third of the intracellular Tfn at 20 min into the new compartment, while the total amount in EEs and REs will remain unaffected . Microscopy failed to detect a third morphologically distinct compartment, and no third peak was detected by subcellular fractionation. However, these techniques might miss a compartment with similar morphology and density characteristics to the RE. Thus, while the presence of a compartment between EEs and REs is unlikely, it cannot be ruled out. Finally, we have made the assumption that the kinetics describing transport between compartments are first order, where the generalized equation is d [ A ]/ dt = − k [ A ], where [ A ] is the concentration of Tfn (or TfnR) in a given compartment and k is the rate constant (see Materials and Methods). Since all of the Tfn is bound to TfnR and under the assay conditions essentially all of the TfnR is occupied by either labeled or unlabeled Tfn, the movement of Tfn– TfnR complexes will be a pseudo first order process. Since these assumptions may not be true, the same model can be constructed using zero order kinetic rates such that d [ A ]/ dt = −( k or [ A ], whichever is lesser). The progress curves produced by such a model are clearly different than the experimentally derived data . Furthermore, a two-compartment, two-pathway zero order model predicts that virtually all of the internalized Tfn will be in the RE even at short times of internalization (2.5 min), clearly at odds with the fluorescence and cell fractionation data. Alternatively, a second order process can be envisioned where both the concentration of Tfn and its receptor play a role such that d [ TfnR ]/ dt = − k [ Tfn ][ TfnR ]. However, since the receptor is essentially fully occupied by the ligand, such a process will reduce a pseudo first order kinetics where d [ Tfn − TfnR ]/ dt = − k [ Tfn − TfnR ]. Still, it is possible there exists some level of cooperativity among the receptors so that the process would still exhibit a second order response to receptor concentration, such that d [ Tfn ]/ dt = − k [ Tfn ] 2 . A model based on second order equations can be made to fit the experimental data . However, the model predicts that in untreated cells, EEs will always contain more Tfn than REs, which is clearly not the case. In epithelial cells, the polarity of receptors and other membrane proteins is determined by a hierarchical arrangement of sorting signals that ensure proper basolateral or apical targeting . Similar or identical signals are decoded in endosomes and the trans-Golgi network. Since receptors such as TfnR are retained almost entirely at the basolateral surface, it must therefore be presumed that recycling is a signal-directed, nonrandom process. However, at least in nonpolarized cells, the apparent rate of TfnR recycling appears indistinguishable from that of bulk membrane lipid, data which have been interpreted to mean that recycling occurs by default . The finding that EEs and REs have distinct sorting features suggests an attractive way to reconcile these apparently disparate ideas. Most recycling from EEs in MDCK cells appears to be directed towards the basolateral surface, and the fidelity of recycling is not affected by AlF 4 . Conceivably, then, there is a nonselective bulk flow of membrane from EEs to the basolateral plasma membrane that might result in the rapid, and possibly even signal-independent, transfer of receptors (TfnR, pIgR) and lipids back to their surface of origin. Those receptors that are transferred to RE, however, may be subjected to a signal-dependent sorting event. Thus, TfnR would be efficiently sorted from pIgR, whose basolateral targeting signal may be inactivated as a result of having bound dIgA . This sorting event presumably reflects the sequestration of TfnR into transport vesicles destined for the basolateral surface while pIgR would be sequestered into apically directed vesicles. AlF 4 may act to prevent the REs from sequestering receptors into such basolateral recycling vesicles. While this information does not bring us much closer to understanding the molecular mechanism of polarized sorting, it does give an important and long awaited hint regarding where to look. | Study | biomedical | en | 0.999996 |
10189374 | The mouse hybridomas producing mAb 9E10 against the c-Myc epitope EQKLISEED or mAb OKT4 to the human CD4 molecule were obtained from the American Type Culture Collection. Mouse mAbs to E-cadherin, calnexin, or caveolin were obtained from Transduction Labs. The anti-amyloid precursor protein antibody was from Boehringer Mannheim . Peroxidase-conjugated secondary anti-Ig antibodies, sulfo- N -hydroxyl-succinimido-biotin (sulfo-NHS-biotin), streptavidin-coupled agarose, and peroxidase-coupled streptavidin were supplied by Pierce. Triton X-100 and octyl-glucoside were purchased from Sigma Chemical Co. Epithelial MDCK II cells from canine kidney were grown on Petri dishes in DME supplemented with 10% of FBS ( GIBCO BRL ), penicillin (50 U/ml), and streptomycin (50 μg/ml) at 37°C in an atmosphere of 5% CO 2 . Influenza virus A/Victoria/3/75 (H3N2) strain (a generous gift from Dr. J. Ortín, Centro Nacional de Biotecnología, Madrid) was grown and titered on MDCK cells. Confluent cell monolayers were incubated with influenza virus (10 pfu/cell) for 1 h at 37°C to allow adsorption and entry of the virus. After that (taken as time 0 of infection), the inoculum was removed and the cell cultures were incubated at 37°C for the indicated times in normal medium. The decapeptide QEGYTYKQYH corresponding to amino acids 114–123 of the dMAL molecule was synthesized on an automated multiple peptide synthesizer (AMS 422; Abimed) using the solid phase procedure and standard Fmoc-chemistry . After coupling to keyhole limpet hemocyanin, the peptide was used to immunize Wistar rats. Spleen cells from immunized rats were fused to myeloma cells following standard protocols , and plated onto microtiter plates. The culture supernatants were screened by immunoblot analysis using GEMs from MDCK cells. The hybridoma clone 2E5 that secretes antibodies to dMAL was isolated after several rounds of screening, and used to produce culture supernatants containing 2E5 mAb. The DNA constructs expressing the hMAL or dMAL proteins tagged at their NH 2 terminus with the 9E10 c-Myc epitope, and the A498 and MDCK stable transfectants expressing tagged hMAL (A498/hMAL and MDCK/hMAL cells) have been described previously . Transient transfection of COS-7 cells with constructs expressing either hMAL or dMAL was carried out by electroporation using Electro Cell Manipulator 600 equipment (BTX). Phosphorothioate oligonucleotides were synthesized with sulfur throughout the phosphate backbone (Isogen Bioscience BV). The 19-mer phosphorothioate oligonucleotide AS (5′-CGCCGCTGCTGGGGCCATG-3′) is complementary to dMAL mRNA, whereas oligonucleotide AM (5′-CGCGGCCACTCGCGTCGTG-3′) is similar in composition to AS but contains some replacements to prevent pairing with dMAL mRNA. Oligonucleotides were introduced into MDCK cells by electroporation. This was carried out in the presence or absence of oligonucleotides AS or AM at 18 μM in 4-mm gap cuvettes by using Electro Cell Manipulator 600 equipment set up at 1.6 kV, 24 Ω, and 50 μF. Under these conditions the actual pulse length obtained in different experiments, as defined by the equipment manufacturer, ranged from 0.5 to 1 ms. Parallel controls to measure the efficiency of the transfection were performed by immunofluorescence analysis with phosphorothioate oligonucleotides labeled with Texas red at the 5′ end. GEMs were isolated by standard procedures . Cells grown to confluency in 100-mm dishes were rinsed with PBS and lysed for 20 min in 1 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 at 4°C. The lysate was scraped from the dishes with a rubber policeman, the dishes rinsed with 1 ml of the same buffer at 4°C, and the lysate homogenized by passing the sample through a 22-gauge needle. The extract was finally brought to 40% sucrose in a final volume of 4 ml and placed at the bottom of an 8-ml 5–30% linear sucrose gradient. Gradients were centrifuged for 18 h at 39,000 rpm at 4°C in a Beckman SW41 rotor. 1-ml fractions were harvested from the bottom of the tube and aliquots were subjected to immunoblot analysis. The method of Skibbens et al. was adopted to analyze the partition of HA into insoluble membranes. In brief, cell monolayers were extracted for 20 min on ice with 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 supplemented with a cocktail of proteases. The extracts were then centrifuged in a refrigerated Hettich microfuge at 14,000 rpm for 1 min. The supernatant (soluble fraction) was removed, and a small amount of the remaining soluble material was recovered from the pellet (insoluble fraction) after a second centrifugation. The soluble material was pooled, and the pellet resuspended in buffer for SDS-PAGE. Finally, equivalent aliquots from the soluble and insoluble fractions were subjected to SDS-PAGE and analyzed by autoradiography or immunoblotting. For immunoblot analysis, samples were subjected to SDS-PAGE in 15% acrylamide gels under reducing conditions and transferred to Immobilon-P membranes ( Millipore ). After blocking with 5% (wt/vol) nonfat dry milk, 0.05% (vol/vol) Tween 20 in PBS, blots were incubated with the indicated primary antibody. After several washings, blots were incubated for 1 h with goat anti–mouse (or anti–rat) IgG antibodies coupled to horseradish peroxidase, washed extensively, and developed using an enhanced chemiluminescence Western blotting kit (ECL; Amersham ). Quantitative analyses were done with the 300A computing densitometer (Molecular Dynamics). For metabolic labeling, cells were starved in culture medium lacking methionine and cysteine for 30 min and incubated with 100–500 μCi of a [ 35 S]methionine/cysteine mixture (ICN) for 10 min at 37°C. After this period, the medium was removed and replaced with standard culture medium. To be used in immunoprecipitation studies, antibodies were prebound overnight at 4°C to protein G–Sepharose in 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100. Cell extracts prepared with 1% Triton X-100 at 37°C or in the presence of 60 mM octyl-glucoside were centrifuged at 14,000 rpm in a microfuge, and the supernatants were incubated for 4 h at 4°C with a control anti-CD4 mAb bound to protein G–Sepharose. After centrifugation, the supernatant was immunoprecipitated by incubation for 4 h at 4°C with mAb 9E10 bound to protein G–Sepharose. The immunoprecipitates were collected, washed six times with 1 ml of 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100, and analyzed by SDS-PAGE under reducing conditions. Immunoprecipitation of surface-biotinylated proteins was carried out with streptavidin-agarose using a protocol similar to that described for immunoprecipitation with antibodies bound to protein G–Sepharose. To detect 35 S-labeling, dried gels were finally exposed to imaging plates (Fuji Photo Film Co.). Quantitative analyses were done with the 300A computing densitometer. For separate access to apical or basolateral domains, MDCK cells were seeded at confluent levels on 24-mm polyester tissue culture inserts of 0.4-μm pore size (Transwell; Costar, Inc.). The integrity of the cell monolayer was monitored by measuring the transepithelial electric resistance using the Millicell ERS apparatus ( Millipore Corp. ). For metabolic labeling of cells in filters, cells infected with influenza virus for 2.5 h were starved in media lacking methionine and cysteine. After 15 min, 250 μCi [ 35 S]methionine/cysteine was added to the basolateral compartment, and filters were incubated for 2 h at 37°C. After repeated washings with ice-cold PBS containing 0.1 mM CaCl 2 and 1 mM MgCl 2 , 0.5 mg/ml sulfo-NHS-biotin were added either to the apical or basolateral compartment of the filter chamber. After 30 min at 4°C, the solution was removed and remaining unreacted biotin quenched by incubation with ice-cold serum-free DME. Cell monolayers were finally washed with PBS and extracted with 0.5 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 60 mM octyl-glucoside for 30 min on ice. Extracts were immunoprecipitated with streptavidin-agarose, and the immunoprecipitates fractionated by SDS-PAGE. To detect the presence of 35 S-labeled HA on the cell surface, blots were exposed to imaging plates. To detect surface E-cadherin, the streptavidin-agarose immunoprecipitates were analyzed by immunoblot with anti–E-cadherin antibodies. We have reported previously the generation of mAb 6D9, which recognizes hMAL but is unreactive to dMAL . We have followed a strategy similar to that used for the generation of anti-hMAL antibodies to produce a novel anti-dMAL antibody. The peptide comprising amino acids 114–123 of dMAL was synthesized and coupled to keyhole limpet hemocyanin. The alignment of the selected canine decapeptide with the corresponding region of hMAL is shown in Fig. 1 A. Spleen cells from Wistar rats immunized with the canine peptide were fused with myeloma cells. During screening of hybridoma culture supernatants for anti-dMAL antibodies, a hybridoma clone (named 2E5) producing antibodies reactive with a protein of the predicted size of MAL (17 kD) was identified by immunoblot analysis of GEM fractions obtained from MDCK cells. To demonstrate that 2E5 mAb does indeed recognize dMAL, tagged forms of hMAL and dMAL were transiently expressed in COS-7 cells. Total cell lysates were prepared 24 h after transfection and subjected to immunoblot analysis with either 6D9, 2E5, or anti-tag 9E10 mAb. Fig. 1 B shows that, whereas mAb 6D9 recognizes hMAL but not dMAL as reported previously , mAb 2E5 is not reactive with hMAL but recognizes dMAL. As a control of the efficiency of the transfections, aliquots from the same samples were analyzed with anti-tag 9E10 mAb. The GEM fraction, which is resistant to solubilization by nonionic detergent at low temperatures, can be separated from the bulk of cellular membranes, which are solubilized by the detergent, and from cytosolic proteins by using an established protocol involving centrifugation to equilibrium on sucrose density gradients . To analyze the distribution of dMAL, MDCK cells were extracted with 1% Triton X-100 at 4°C, and the extracts were centrifuged to equilibrium. 12 1-ml fractions were obtained after fractionation of the gradient from the bottom of the tube. When the different fractions were analyzed by immunoblotting with anti-MAL 2E5 mAb, MAL was detected exclusively as being present in the floating detergent-resistant membrane fractions, indicating that endogenous MAL specifically resides in GEMs in MDCK cells . The distribution of caveolin and calnexin along the gradient, as respective representatives of proteins included or excluded from GEMs, are shown as internal controls of the fractionation procedure. To approach directly the possible role of MAL in apical transport, we designed a 19-mer phosphorothionate oligonucleotide complementary to the sequence surrounding the AUG translation initiation site of dMAL mRNA (oligonucleotide AS) and a control oligonucleotide (oligonucleotide AM) with a composition similar to that of AS, but differing from it in seven nucleotides scattered along its sequence . These oligonucleotides were transfected by electroporation into MDCK cells and 48 h later, MDCK cell extracts were analyzed by immunoblotting with mAb 2E5. Fig. 3 B shows that whereas the control oligonucleotide AM did not affect the levels of MAL, transfection of oligonucleotide AS greatly diminished the amount of endogenous MAL in MDCK cells. The extent of the depletion varied between experiments, probably due to differences in the actual pulse length set up by the electroporation device in each transfection. Parallel experiments using Texas red–labeled phosphorothioate oligonucleotides indicated that the efficiency of transfection varied between 80 and 99% of the cells as assayed by immunofluorescence analysis (not shown). The MAL levels obtained in cells electroporated with oligonucleotide AS were usually 10–80% of the amount of MAL found in cells electroporated with oligonucleotide AM. The levels of caveolin and that of the amyloid precursor protein , two proteins found in GEMs in MDCK cells, were not affected by this treatment . When the effect of oligonucleotide AS was assayed on MDCK cells ectopically expressing a tagged form of hMAL, we found that whereas the endogenous dMAL protein was depleted, the levels of the exogenously expressed protein were almost completely unaffected . This is probably due to the fact that both the insertion of sequences encoding the c-Myc 9E10 epitope after the translation initiation codon, and the presence of a base change in the human sequence in the region covered by oligonucleotide AS, reduce the number of nucleotides that pair with the ectopic hMAL mRNA species . In addition, the presence of the c-Myc epitope-encoding sequences separates oligonucleotide AS from the translation initiation site, which is generally considered to be one of the best target sites for obtaining good depletions with antisense oligonucleotides . Taking into account the previously proposed role of MAL in raft organization , we first investigated whether or not MAL levels affect the presence of HA in GEMs as measured by insolubility in 1% Triton X-100 at 4°C using the sedimentation procedure described by Skibbens et al. . MDCK cells were transfected with oligonucleotides AM or AS, and infected 48 h later with influenza virus. After 2.5 h, cells were labeled with [ 35 S]methionine/cysteine for 10 min, and chased for 2 h in normal medium. The soluble and insoluble fractions were separated by centrifugation in a microfuge after extraction of the cells with 1% Triton X-100 at 4°C, and were subjected to SDS-PAGE and autoradiographed. Viral proteins were easily identified due to the profound shut-off of host protein synthesis induced by influenza virus infection (not shown). For simplicity, only the band corresponding to HA is shown in the experiments presented here. The extent of MAL depletion was quantified by densitometric analysis of immunoblots of the initial lysates from cells transfected with either oligonucleotide AM or AS with mAb 2E5, and the partition of radiolabeled HA into the soluble and insoluble fractions was measured by densitometric analysis of the autoradiograms. Fig. 4 B shows the partition of HA in cells with the indicated levels of endogenous MAL. Fig. 4 A shows, as an example of those experiments, the partition of HA in the soluble and insoluble fractions under conditions in which the endogenous MAL levels were ∼15% of those in control cells. As shown in Fig. 4 , A and B, HA insolubility decreased progressively with the reduction of MAL levels, indicating that MAL might favor HA residence within GEMs. The insolubilities of caveolin and amyloid precursor protein were not affected by MAL depletion (not shown). To further address the role of MAL in HA insolubility we carried out pulse-chase experiments to analyze the incorporation of HA into GEMs in A498 cells, which lack endogenous MAL expression and in A498/hMAL cells, which express ectopically tagged hMAL. Fig. 4 , C and D, shows that although HA became progressively insoluble in normal A498 cells, the expression of MAL in these cells caused a marked increase in the insolubility of HA at all the chase times assayed. Furthermore, the effect of MAL expression in HA insolubility was not due to an artifact of the sedimentation procedure caused by differences in size of the Triton X-100–resistant membranes between A498 and A498/hMAL cells, as it also was observed when HA insolubility was assayed by floatation using centrifugation to equilibrium . To examine whether there is an association between HA and MAL during transport of newly synthesized HA to the cell surface, we carried out immunoprecipitation experiments in MDCK cells infected with influenza virus. As anti-MAL 2E5 mAb works poorly in immunoprecipitation analysis, this study was performed using anti-tag mAb 9E10 and MDCK cells stably expressing tagged hMAL (MDCK/hMAL cells). Cells were infected with influenza virus, incubated at 37°C for 2.5 h, labeled for 1 h with [ 35 S]methionine/cysteine, and maintained at 20°C for 1 h to accumulate radiolabeled HA in the trans-Golgi network . Finally, cells were extracted with 1% Triton X-100 at either 4 or 37°C, or with 1% Triton X-100 plus 60 mM octyl-glucoside. Fig. 5 A shows that, in contrast with the extract prepared at 4°C, both HA and MAL are in the soluble fraction (supernatant) in the extract prepared at 37°C as assayed by the sedimentation procedure , in agreement with previous reports showing that treatment at 37°C solubilizes proteins in GEMs . To rule out the possibility that the presence of HA and MAL in the supernatant was due to differences in the size of remaining membrane complexes resistant to extraction at 37°C, the supernatant fraction was subjected to centrifugation to equilibrium in sucrose density gradients. Fig. 5 B shows that under those conditions of extraction both HA and MAL appear exclusively in the fractions containing soluble material. The supernatant from the 1% Triton X-100 extract prepared at 37°C was subsequently subjected to immunoprecipitation with anti-CD4 mAb OKT4, taken as control mAb, or with anti-tag mAb 9E10. The presence of tagged-hMAL in the immunoprecipitates was assayed by immunoblotting with mAb 9E10 and that of HA by autoradiography. The right panel of Fig. 5 C shows that HA and MAL coimmunoprecipitate in the extracts prepared at 37°C. Furthermore, this association is not the result of a redistribution of the proteins during the extraction procedure as revealed by the absence of HA in the immunoprecipitates obtained using a similar extract prepared using a mixture of 35 S-labeled wild-type (untransfected) MDCK cells infected with influenza virus and uninfected MDCK/hMAL cells . The possibility of an unspecific interaction of mAb 9E10 with HA was ruled out by the absence of HA in the 9E10 mAb immunoprecipitates obtained using extracts from untransfected MDCK cells infected with influenza virus . Extraction with octyl-glucoside is a second procedure widely used to solubilize proteins in GEMs . When MDCK/hMAL cells infected with influenza virus were extracted with octyl-glucoside, both HA and MAL appeared in the soluble fraction . Immunoprecipitation of tagged-MAL with mAb 9E10 revealed that, under these conditions of solubilization, the association of HA with MAL is lost . The effect of MAL depletion on HA insolubility and the observed association of MAL with HA during HA biosynthetic transport led us to analyze whether MAL depletion affects the delivery of HA to the cell surface. MDCK cells were transfected with oligonucleotides AM or AS, and infected 48 h later with influenza virus. After 1 h at 37°C to allow virus adsorption and entry, cells were incubated for 2.5 h and then labeled with [ 35 S]methionine/cysteine for 10 min. Cells were chased for 0, 30, or 60 min in normal medium. Surface proteins were labeled with biotin at the end of each chase period, and biotinylated proteins were immunoprecipitated with streptavidin-agarose. The rate of arrival of newly synthesized radiolabeled HA at the cell surface was determined by autoradiography of the immunoprecipitates. Fig. 6 , A and C, shows that although HA radiolabeling was similar in both cases (not shown), the kinetics of HA delivery to the plasma membrane was faster in cells transfected with control oligonucleotide AM than in cells in which MAL levels were depleted by transfection of oligonucleotide AS. When we compared the kinetics of surface arrival of HA in normal A498 cells and A498/hMAL cells, we observed that, consistent with the hypothesis of a role for MAL in HA transport, the ectopic expression of MAL in A498 cells enhanced the rate of delivery of HA to the cell surface . GEM disruption by lowering cell cholesterol produces reduction of apical transport and partial missorting of HA to the basolateral membrane . To examine whether MAL depletion causes a similar effect on HA transport, we compared the apical and basolateral delivery of HA in MDCK cells with either normal or depleted levels of MAL. To this end, cells were transfected with either oligonucleotides AM or AS, and seeded at high density (3.5–5.0 × 10 5 cells/cm 2 ) on filter culture inserts. After 48 h at 37°C, the integrity of the cell monolayers was checked, and intact cell monolayers were infected with influenza virus. 2.5 h after removal of the inoculum, newly synthesized proteins were labeled with [ 35 S]methionine/cysteine for 2 h. Surface proteins were then separately biotinylated from the apical or basolateral face, and immunoprecipitated with streptavidin-agarose. The apical or basolateral surface expression of HA was determined by autoradiography of the corresponding streptavidin-agarose immunoprecipitate. As an internal control, the sorting of E-cadherin, a basolateral protein , was determined by immunoblot analysis with anti–E-cadherin antibodies of the streptavidin-agarose immunoprecipitates. The extent of MAL depletion obtained in each experiment was quantified by densitometric scanning of immunoblots of the initial lysates with anti-dMAL 2E5 mAb. A representative experiment in which MAL levels dropped to ∼12% of those in control cells is shown in Fig. 7 A. Whereas only 10% of surface HA was on the basolateral membrane in control MDCK cells, missorting to this domain increased to ∼70% in the cells with reduced MAL levels. To confirm that the observed effects were due to MAL depletion and not to spurious effects of the AS oligonucleotide, we took advantage of the selectivity of oligonucleotide AS in blocking expression of endogenous dMAL but not of tagged hMAL in MDCK/hMAL cells to demonstrate whether ectopic expression of MAL rescues the effects observed in MAL-depleted MDCK cells. The right panel of Fig. 7 A shows that the expression of exogenous hMAL restored apical transport of HA, and prevented HA missorting to the basolateral membrane, in spite of the drop of endogenous MAL to ∼12% of that in normal cells. HA insolubility was also rescued by exogenous hMAL in MDCK cells with depleted levels of the endogenous protein (not shown). A compilation of the results obtained on HA transport to the apical or basolateral membranes from experiments in which MAL was depleted to different extents is shown in Fig. 7 B. Quantitative analysis of the amount of HA on the apical or the basolateral domains indicates that the altered ratio of apical to basolateral HA targeting observed upon MAL depletion was not only due to decreased HA transport to the apical surface but also to a concomitant increased transport of HA to the basolateral surface . Moreover, consistent with the kinetics of HA arrival at the plasma membrane in MDCK cells , the total (apical + basolateral) delivery of HA to the cell surface was reduced in cells with depleted levels of MAL . In summary, Fig. 7 indicates clear correlations between MAL depletion, progressive reduction of apical HA sorting, and concomitant increased transport of HA to the basolateral membrane. Transport of influenza virus HA to the apical membrane should be dependent on the lipid components of GEMs and the protein elements of the apical sorting machinery. Lowering the cellular cholesterol levels increases the solubility of HA in Triton X-100. This indicates that cholesterol is essential for association of HA with GEMs . Using an antisense approach to decrease the level of MAL in MDCK cells, we have found that MAL is also required for normal incorporation of HA into GEMs. Caveolin and amyloid precursor protein, found in GEMs from caveolae or from a protein processing compartment , were unaffected by MAL depletion, indicating that the observed effect was specific for HA, and that MAL depletion does not affect the insolubility of proteins sequestered in other types of GEM rafts. This might be interpreted as meaning that although HA does not need MAL to gain access into GEMs, the presence of MAL favors the interaction of HA with rafts. This interpretation is supported by the following observations. In A498 cells, which lack endogenous MAL expression, the levels of HA in GEMs were lower than those in MDCK cells. Ectopic expression of MAL in A498 cells caused a dramatic increase in the levels of HA in GEMs. Thus, it is plausible that HA gains access into GEMs by itself, requiring specific residues in its transmembrane domain , and that the presence of MAL helps the incorporation of HA into GEMs either through a direct interaction with HA or, indirectly, by affecting the GEM rafts. GEM-dependent transport of influenza virus HA involves incorporation of HA into GEMs, formation of transport vesicles, and targeting and delivery of HA to the apical membrane. Based on the behavior of a panel of HA mutants, a multistep process for the initial steps of apical transport has been postulated . According to this model, at least some of the integral membrane components of the sorting machinery acting in the GEM-mediated pathway of apical transport are totally sequestered in GEMs. These elements would come into contact with integral proteins capable of access into GEM rafts, and only proteins able to interact with the machinery would be efficiently concentrated and effectively sorted to the apical surface. Using the newly generated 2E5 mAb to dMAL protein, we have demonstrated that endogenous MAL is exclusively confined to GEM microdomains in MDCK cells. Moreover, using MDCK cells infected with influenza virus we have found that HA associates with MAL during biosynthetic transport. This association is preserved when the extracts are prepared with 1% Triton X-100 at 37°C but is lost in the presence of octyl-glucoside. The effect of octyl-glucoside indicates that either HA and MAL are not directly associated or simply that, similar to protein–lipid interactions in GEMs, their direct protein–protein interaction is sensitive to octyl-glucoside. Our results showing coimmunoprecipitation of HA and MAL in Triton X-100 extracts prepared at 37°C indicate that if there are still any lipids maintaining the association between these two proteins, those lipids are not sufficient to provide buoyancy to the HA–MAL complex as assayed by centrifugation to equilibrium or to make the complex sedimentable under conditions used to sediment GEMs. The decrease in the insolubility of HA in MAL-depleted MDCK cells correlated with a partial inhibition on HA transport to the plasma membrane. This is consistent with recent findings showing that the rate of arrival of HA at the plasma membrane was diminished in cells in which HA insolubility was lowered by removal of cholesterol . This indicates that both cholesterol and MAL are required for normal transport of HA to the cell surface. As evidence for the role of MAL in HA transport, we have found that the enhanced incorporation of HA in GEMs by ectopic expression of hMAL in A498 cells is accompanied by an increase in the rate of transport of HA to the cell surface. As HA is delivered to the plasma membrane in cells that lack MAL expression (e.g., wild-type A498 cells), it is obvious that MAL is not strictly necessary for transport of HA to the cell surface. Rather, we speculate that MAL is only necessary for a specialized route of transport involving GEMs that takes place only in a restricted range of cell types. The proposed roles of MAL GEM-mediated transport led us to examine the obvious possibility that MAL is an element of the apical transport machinery in MDCK cells. As predicted, MDCK cells with reduced MAL levels showed a lower HA transport to the apical surface compared with that in control MDCK cells. The loss of apical HA transport was partially compensated for by the appearance of HA at the basolateral surface. These results resemble those obtained in MDCK cells with GEM rafts disrupted by cholesterol depletion or with HA mutants unable to access GEMs in which HA was also partially missorted to the basolateral membrane. Thus, although HA is normally excluded from basolateral vesicles, it appears that the lack of stabilization of HA into GEMs produced by MAL removal induced partial incorporation of HA into vesicles destined for the basolateral surface. The fact that ectopic expression of hMAL prevented the effect of MAL depletion on HA insolubility and sorting strongly argues against an artifactual effect of oligonucleotide AS in HA transport. GEMs have been isolated from a variety of tissues, and their existence seems to represent a general feature influencing the function of cellular membranes . The MAL protein has been identified as a component of GEMs in all of the cell types in which it is expressed . The fact that MAL gene expression is tissue- and differentiation-specific suggests that the role of MAL in GEMs is required in only a few cell types, and that this role is probably related to a specialized function common to this restricted range of cells. In polarized epithelial cells, endogenous MAL is localized to the apical zone, consistent with its proposed role in apical transport . Based on the extensive vesiculation induced by ectopic expression of MAL in Sf21 insect cells, MAL was proposed as being an element of the apical machinery responsible for GEM vesiculation . The vesicles induced by MAL expression in insect cells were clearly different from the caveolae-like vesicles induced by caveolin as shown by coexpression of the two proteins. Thus, it was proposed that both caveolin and MAL may belong to the vesicular transport machinery specific for GEMs but acting in the assembly of different classes of GEM microdomains . Caveolin appears to be related to caveolae formation , whereas our present results support the hypothesis of the involvement of MAL in apical transport. The GEM-based model for sorting of apical proteins predicts the existence of sorting receptors responsible for recruiting cargo proteins . The results presented in this study show that: HA associates with MAL in GEMs during biosynthetic transport; the presence of MAL favors the incorporation of HA into GEMs; and MAL is necessary for normal apical transport and accurate sorting of HA. Thus, in addition to the proposed roles of MAL in formation of vesicles for GEM-dependent transport and GEM trafficking , MAL also plays a role in the confinement of influenza virus HA into GEMs and in its transport to the apical surface. | Other | biomedical | en | 0.999997 |
10189375 | SPN cDNA clones were isolated by hybridization screening of a CLONTECH rabbit skeletal muscle cDNA library with a PCR-derived SPN cDNA probe encoding exons 1–3. Sequence analysis of the clones was performed using dye terminator cycling and analyzed on a 373 stretch fluorescent automated sequencer (PE Applied Biosystems). The nucleotide and deduced amino acid sequences of rabbit SPN have been deposited in the GenBank/EMBL/DDBJ data bank with the accession number AF120276 . Multiple sequence alignment was performed using the DNAsis sequence analysis software (Hitachi Software Engineering, Inc.). Also, we have isolated SPN cDNA clones independently from a mouse skeletal muscle library and found clones identical to those found by Scott et al. . Adult mouse multiple tissue Northern blots ( CLONTECH Laboratories, Inc.) containing 2 μg of poly (A) + RNA per lane were probed with an expressed sequence tag corresponding to the 3′ untranslated region of mouse SPN . Identical results were obtained when blots were hybridized with PCR-amplified probes representing the entire coding region of mouse SPN. Wild-type (wt; C57BL/10) and mdx (C57BL/10ScSn) mice, obtained from Jackson ImmunoResearch Laboratories, Inc. were maintained at the University of Iowa Animal Care Unit in accordance with animal usage guidelines. The dystrophin transgenic mice have been described previously . Male F1B and BIO 14.6 cardiomyopathic hamsters were obtained from BioBreeders. We have previously reported the generation and initial characterization of the α-SG deficient (Sgca-null) mice . The targeted disruption of the α-SG gene was accomplished by replacement of exons 2 and 3, and flanking intronic sequences with the neomycin resistance gene through homologous recombination . Utrophin deficient (utrn −/− ) and utrophin–dystrophin deficient mice ( mdx :utrn −/− ) have been described previously . Utrn −/− and mdx :utrn −/− mice were maintained at Washington University (St. Louis, MO). mAbs against α- (20A6), β- (5B1), and γ-SG (21B5), as well as mAbs against β-DG (8D5) were generated in collaboration with Dr. Louise V.B. Anderson (Newcastle General Hospital, Newcastle upon Tyne, UK). mAb against α-DG (IIH6) have been described by Ervasti and Campbell . Antibodies against the laminin α2 chain and the NH 2 terminus of rabbit SPN have been described previously. For generating antibodies against mouse SPN, two New Zealand White rabbits (rabbits 235 and 236; Knapp Creek Farms) were injected at intramuscular and subcutaneous sites with a COOH-terminal SPN–glutathione S transferase fusion protein (amino acids 186–216 of mouse SPN; CFVMWKHRYQVFYVGVGLRSLMASDGQLPKA). Affinity purification of SPN antibodies was accomplished using Immobilon-P ( Millipore Corp. ) strips containing the COOH-terminal SPN–maltose-binding fusion protein. Antibody specificity was verified for both immunofluorescence and immunoblotting by competition experiments using the COOH-terminal SPN fusion protein and peptides synthesized to the COOH-terminal region of mouse SPN (data not shown). Transverse muscle cryosections (7 μm) were analyzed by immunofluorescence as described in Crosbie et al. . For extraocular muscle (EOM) studies, rectus muscles (global layer) were examined. Affinity purified rabbit 235 SPN antibody was incubated at a dilution of 1:50 and 1:10 with mouse and hamster sections, respectively. After washing with TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.4), the sections were incubated with Cy3-conjugated secondary antibodies at a dilution of 1:250 (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. For staining of neuromuscular junctions (NMJs), samples were simultaneously incubated with fluorescein-conjugated α-bungarotoxin (1:1,000; Molecular Probes, Inc.). After washing with TBS, the slides were mounted with Vectashield mounting medium (Vector Labs Inc.) and observed under a BioRad MRC-600 laser scanning confocal microscope. Digitized images were captured under identical conditions. The human δ-SG cDNA sequence was subcloned into the pAdRSVpA adenovirus vector through standard methods of homologous recombination with Ad5 backbone dl309 by the University of Iowa Gene Transfer Vector Core. Preparation of the recombinant adenovirus and the intramuscular injections were performed as previously described . In brief, 10 9 viral particles in 100 μl of normal saline were injected into the quadriceps femoris of 3-wk-old BIO 14.6 hamsters after the animals were anesthetized by intraperitoneal injection of sodium pentobartital (Nembutal; Abbott Laboratories) at a calculated dose of 75 mg/kg. Quadriceps muscle was collected 2 wk after the injection. KCl washed membranes from wt, mdx , and Sgca-null mice were prepared from skeletal muscle as described previously . Purified DGC from rabbit skeletal muscle membranes was titrated to pH 11 using 1 M NaOH and incubated for 1 h at room temperature with gentle mixing in a buffer consisting of 50 mM Tris, 0.1% digitonin, 175 mM NaCl, 0.1 mM PMSF, 0.75 mM benzamidine. The alkaline treated DGC was concentrated fourfold using Centricon-10 filters (Amicon Corp.). The samples were loaded onto 5–30% linear sucrose gradients in a buffer of 50 mM Tris-HCL, 500 mM NaCl, 0.1% digitonin, 0.1 mM PMSF, 0.75 mM benzamidine, pH 11. The gradients were centrifuged at 4°C in a Beckman Vti 65.1 vertical rotor for 2.5 h at 200,000 g . 16 0.8-ml fractions were collected from the top of the gradient using an Isco model 640 density gradient fractionator. The protein samples (60 μl) were separated by 3–15% SDS-PAGE and immunoblotted, as described (vide infra). Quadriceps femoris muscle was dissected from mdx mice and snap frozen in liquid nitrogen. Frozen tissue (1 g) was pulverized into small pieces with a pestal and mortar filled with liquid nitrogen. The tissue was solubilized by dounce homogenization in 10 ml of cold buffer A (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 1.0% digitonin) with a cocktail of protease inhibitors (0.6 μg/ml pepstatin A, 0.5 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.1 mM PMSF, 0.75 mM benzamidine, 5 μm calpain I inhibitor, and 5 μM calpeptin). The samples were spun at 142,400 g for 37 min at 4°C. The pellets were resolublized with 5 ml of buffer A, rotated at 4°C for 1 h, and centrifuged as before. The two supernatants were combined and incubated overnight at 4°C with 1 ml of WGA–Sepharose (Vector Labs, Inc.). The WGA–Sepharose was washed extensively (50 mM Tris-HCl, pH 7.8, 0.1% digitonin, 500 mM NaCl) and proteins were eluted with 0.3 M N-acetyl glucosamine ( Sigma Chemical Co. ). Samples were concentrated to 500 μl using a Centricon-30 filter and applied to a 5–30% sucrose gradient at pH 7.8, as described previously . Protein samples were resolved under reducing conditions by 3–15% SDS-PAGE and transferred to PVDF (Immobilon-P) membranes ( Millipore Corp. ). PVDF membranes were probed with anti-SG mAbs, as described previously . For mouse SPN immunoblotting, the membranes were probed with affinity purified rabbit 235 antibody at a dilution of 1:50. Note that for mouse SPN immunoblotting, proteins were resolved on 3–15% SDS-PAGE under nonreducing conditions and transferred to PVDF (Immobilon-P). For rabbit SPN staining, nitrocellulose blots were probed with affinity purified rabbit 216 antibody as described . For α- and β-DG staining, SDS-polyacrylamide gels were transferred to nitrocellulose (Immobilon-NC) and probed with IIH6 (1:3 dilution) and 20A6 (1:100 dilution). Following incubation with primary antibodies, blots were probed with the appropriate HRP-conjugated secondary antibodies (1:5,000; Boehringer Mannheim Corp. ) and developed using enhanced chemiluminescence (SuperSignal; Pierce Chemical Co. ). A human SPN expression construct was prepared by PCR amplification of cDNA using primers containing appropriate restriction sites for subcloning into pcDNA3 ( Pharmacia Biotech, Inc. ). The SPN construct was engineered to encode a myc-tag at the COOH terminus. All constructs were verified by direct DNA sequence analysis performed by the DNA Core Facility at the University of Iowa (Iowa City, IA). Full-length myc-tagged α-, β-, γ-, and δ-SG pcDNA3 ( Pharmacia Biotech Inc. ) expression constructs have been previously described . Construction and design of the Grb2 cDNA expression vector has been described . CHO cells were electroporated with SG and SPN expression constructs (∼5 μg of each plasmid DNA) at 340 V at 950 μF using a BioRad electroporator, as previously described . 30 h after transfection, cells were analyzed for protein expression by SDS-PAGE and immunoblotting. Membrane surface proteins were biotinylated using membrane impermeant sulfo-NHS-biotin ( Pierce Chemical Co. ) as described previously for the SGs expressed in CHO cells . Immunoprecipitation using a β-SG mAb (5B1) and analysis of protein samples by SDS-PAGE and immunoblotting with an anti-myc mAb (9E10) were performed as documented in Holt and Campbell . SPN is the most recently identified dystrophin-associated protein, and therefore the least characterized. Hydropathy analysis of the primary amino acid sequences of human and murine SPN predicts a protein with intracellular NH 2 and COOH termini, and four transmembrane domains. We report determination of the primary structure of rabbit SPN, as deduced from a rabbit skeletal muscle cDNA . Multiple sequence alignment demonstrates that amino acid sequences derived from rabbit, mouse, and human SPN are ≥75% identical . Human and rabbit SPN contain a short insertion at the NH 2 terminus, which is absent in mouse SPN. The four predicted transmembrane domains are extremely well conserved. SPN's membrane topology is strikingly different from other dystrophin-associated proteins, which only have a single pass transmembrane domain, and is reminiscent of the tetraspan superfamily of proteins . Using phylogenetic analysis, we previously demonstrated that SPN is closely related to the divergent family members Rom-1, peripherin, and uroplakin . The tetraspans are thought to play important roles in mediating interactions between transmembrane proteins as mechanisms to control cell growth and adhesion. We speculate that SPN, a novel dystrophin-associated tetraspan, may be facilitating interactions among proteins of the DGC and perhaps mediating interactions of the DGC components with other sarcolemma proteins. To examine the distribution of SPN in mouse tissue, we performed RNA hybridization analysis. Hybridization of mouse multiple tissue Northern blots with probes representing either the coding region or the 3′ untranslated region of SPN gave identical results . As shown in Fig. 1 , a 4.4-kb transcript is predominant in skeletal and cardiac tissues, with minor transcript levels present in lung, brain, and testis. Human skeletal and cardiac muscles express two SPN transcripts . The additional 6.5-kb transcript in humans is likely the result of alternate splicing, since the 6.5-kb transcript does not hybridize to SPN exons 2 and 3 . We have previously shown that SPN is expressed throughout the sarcolemma of normal human skeletal muscle . The limitations of patient biopsies with known mutations prompted our use of murine models as a method to investigate the interactions of SPN with the DGC. First, we examined expression of SPN in quadriceps femoris (thigh), diaphragm, and cardiac muscles from mdx mice, a model for Duchenne muscular dystrophy. The mdx phenotype is inherited as an X-linked recessive trait, which stems from a premature stop codon in exon 23 of the dystrophin gene, leading to absence of dystrophin protein . As a consequence, the dystrophin-associated proteins are nearly ablated from the sarcolemma . Using indirect immunofluorescence on muscle cross sections, we show that SPN is dramatically reduced in skeletal muscle of mdx mice . Our data also demonstrate that SPN is expressed in normal cardiac tissue, which is consistent with the presence of SPN transcript in Northern blots . As shown in Fig. 2 , SPN is significantly reduced in mdx cardiac tissue. The diaphragm muscle, which is the most severely affected muscle in mdx mice, also lacks normal sarcolemma expression of SPN. In addition to its expression in skeletal muscle, recent experiments from our group demonstrate that SPN is also present in smooth muscle (Straub, V., and K.P. Campbell, personal communication). Lastly, we observed positive SPN staining in muscle from the laminin α2 deficient dy and dy 2J mice (data not shown), which are naturally occurring animal models of congenital muscular dystrophy. To further explore the association of SPN with the DGC, we examined SPN expression in a collection of dystrophin transgenic mice. The transgenes, which were individually expressed on an mdx background, encode truncated dystrophin products. Muscle from the transgenic mice was previously evaluated for its morphology and for the ability of the transgene to restore sarcolemma localization of the DGC . We detected SPN expression in skeletal muscle cross sections by indirect immunofluorescence staining with SPN antibodies, and found normal levels of SPN expression in muscle from Δ17–48 , Δ1–62 , Δ71–74 , and Δ75–78 transgenic mice . Despite large deletions, these transgenes are able to restore SPN to the sarcolemma and, with the exception of the Δ1–62 transgene, alleviate muscular dystrophy. The Δ71–74 transgene represents an alternately spliced dystrophin isoform that is predominantly expressed in brain, and the Δ1–62 (Dp71) transgene mimics a form of dystrophin present in lung, spleen, testis, and retina. To determine if SPN is associated with the utrophin–glycoprotein complex, and if replacement of dystrophin with utrophin would affect SPN's localization to the sarcolemma, we examined NMJs from dystrophin, utrophin , and dystrophin–utrophin deficient muscle. The NMJs were identified by staining cross sections of quadriceps femoris with fluorescein α-bungarotoxin, which selectively binds to acetylcholine receptors. By indirect immunofluorescence, we show that SPN is enriched at the NMJ of innervated muscle . This enrichment is maintained even after denervation, demonstrating that SPN is associated with the postsynaptic membrane (data not shown). At the NMJ, dystrophin is replaced by the structurally and functionally similar protein, utrophin . Enrichment of SPN at the NMJ is not altered by the absence of dystrophin, as seen by positive NMJ staining in the mdx muscle . In this case, SPN's localization to the NMJ is mediated by utrophin. Conversely, NMJ localization of SPN is preserved by dystrophin in utrn −/− muscle, as demonstrated by SPN NMJ staining in these mice. Loss of SPN staining from the NMJ occurs only in the absence of both utrophin and dystrophin, as in the mdx : utrn −/− double mutant mice. In mdx mice, muscles with the greatest upregulation of utrophin exhibit the least pathological changes . For instance, the EOM are spared the pathological effects in mdx mice and Duchenne muscular dystrophy patients, likely from the upregulation of utrophin . In support of this, Tinsley et al. demonstrate that expression of utrophin attenuates the dystrophic pathology in mdx mice, suggesting that utrophin can functionally replace dystrophin within the complex. We examined the EOMs from wt, mdx , and mdx :utrn −/− mice for SPN expression as another method to demonstrate that SPN is part of the utrophin–glycoprotein complex. We show that SPN is located at the sarcolemma of wt EOM and is maintained in the EOM of mdx mice, despite absence of dystrophin . The continued expression of SPN in the mdx EOM likely is mediated through SPN's association with the utrophin–glycoprotein complex. Consistent with this idea, SPN expression is lost in the EOM of mice lacking both dystrophin and utrophin . These data are important as they demonstrate that upregulation of utrophin retains SPN to the sarcolemma and validates this as a reasonable therapy for Duchenne muscular dystrophy. The SGs (consisting of α, β, γ, and δ subunits) form a tight subcomplex of four transmembrane glycoproteins within the DGC . The integrity of this complex is maintained despite harsh treatments with SDS and n -octyl β- d -glucoside . Absence of any one of the SGs results in absence of the entire SG subcomplex and destabilization of α-DG from the sarcolemma . Furthermore, this subcomplex is critical for protecting the sarcolemma from contraction induced damage. We wanted to determine if SPN depends on the SG subcomplex for proper membrane targeting by examining δ-SG-deficient BIO 14.6 hamsters for SPN expression. A large deletion in the δ-SG gene causes selective loss of the entire SG subcomplex from BIO 14.6 skeletal muscle without affecting β-DG . We now demonstrate that SPN expression is absent from the sarcolemma , as well as the NMJ (data not shown) of the BIO 14.6 hamster. Furthermore, we show that SPN expression is restored to normal levels after delivery of an adenovirus encoding δ-SG into BIO 14.6 muscle . Control injections of α-SG did not restore proper localization of SPN or the SGs . Recent experiments from our laboratory have shown that injection of δ-SG into muscle of the BIO 14.6 hamster rescues expression of the entire SG subcomplex . Muscle fibers expressing the restored SG–SPN subcomplex are spared the pathological features of muscular dystrophy (i.e., sarcolemma damage and central nucleation) and have stable expression of α-DG at the plasma membrane . Thus, SPN and the SGs are required for normal muscle physiology and prevention of dystrophic features. In addition to this naturally occurring hamster model for LGMD, our laboratory has created α-SG null mice by a targeted disruption of the murine α-SG gene . Like the BIO 14.6 hamsters, Sgca-null mice specifically lack the SG subcomplex . We now demonstrate that Sgca-null muscle is completely devoid of SPN . The NMJ and myotendinous junction (MTJ), which we show are normally enriched for SPN expression, also lack SPN in the Sgca-null mice . As further demonstration of the tight association of SPN with the SGs, we immunoblotted KCl washed membranes prepared from skeletal muscle of wt, mdx , and Sgca-null mice. SPN is dramatically reduced in mdx membranes (∼90% compared with wt), but SPN was not detected in the Sgca-null membranes . To demonstrate that SPN is tightly associated with the SGs, we isolated the SG–SPN subcomplex from skeletal muscle. We prepared purified DGC from rabbit skeletal muscle microsomes and titrated the complex to pH 11 to dissociate pH-sensitive protein–protein interactions. Alkaline-treated DGC was centrifuged through a 5–30% linear sucrose gradient. Proteins from the sucrose gradient fractions were separated by SDS-PAGE and immunoblotted with anti-DGC antibodies. As shown in Fig. 8 a, sucrose gradient sedimentation of alkaline-treated DGC separates the DG (fractions 6–9) and SG (fractions 9–12) subcomplexes from one another. SPN displays a sedimentation pattern similar to that of the SG subcomplex, indicating a preferential association of SPN with the SGs. In addition to chemically disrupting the DGC, we analyzed the dissociation of SG and DG subcomplexes resulting from the absence of dystrophin. The dystrophin-associated proteins are present in the extrajunctional sarcolemma of mdx muscle, although at significantly reduced levels. Figs. 2 and 7 illustrate that ∼10% of SPN expression is maintained at the mdx sarcolemma. We prepared glycoproteins by WGA–Sepharose chromatography of digitonin-solubilized mdx skeletal muscle. Without dystrophin, the SG and DG subcomplexes are no longer associated and can be separated by sucrose gradient centrifugation. The subcomplexes peak in separate fractions and the relative separations between the SG and DG containing fractions are similar for both mdx and pH 11 treated samples. As shown in Fig. 8 b, SPN migrates exclusively with the SG containing fractions. Using an in vivo cell expression system, we demonstrate that SPN and the SGs are associated in a complex at the plasma membrane. Myc-tagged human cDNA constructs of the SGs (α, β, γ, and δ) and SPN were transiently introduced into CHO cells by electroporation. Immunoblots of cellular protein lysates with anti-myc antibodies demonstrate that each of the SGs and SPN, as well as the Grb2 negative control, are expressed at relatively equal quantities . We confirm that these proteins are targeted to the plasma membrane by treatment of cells with sulfo-NHS-biotin, which forms a covalent bond with free amines of proteins at the cell surface. Clarified lysates from transfected CHO cells were incubated with avidin–Sepharose to precipitate plasma membrane–associated proteins. As shown in Fig. 9 , SPN and the SGs are properly localized to the plasma membrane. To demonstrate that the SGs and SPN are assembled into a stable molecular complex, we performed immunoprecipitation experiments. CHO cells transfected with the SGs plus SPN were immunoprecipitated using mAbs to β-SG. SPN coimmunoprecipitates along with the SGs from CHO cells. To demonstrate the specificity of this association, control immunoprecipitation experiments from cells expressing the SGs and myc-tagged Grb2 were performed. Grb2 serves as a negative control since it is a soluble protein that is not expected to associate with the SGs at the plasma membrane. The SGs and Grb2 were cotransfected into CHO cells and cellular lysates were immunoprecipitated with the β-SG mAb. Grb2 is not found in the immune complex with the SGs . These data provide strong evidence that the simultaneous expression of the SGs and SPN in CHO cells results in the formation of a tight molecular complex. The DGC spans the sarcolemma and links the intracellular actin cytoskeleton of muscle cells to the extracellular matrix. Current evidence indicates that the DGC confers structural stability to the muscle plasma membrane, thus protecting it from stresses that develop during muscle fiber contraction. In support of this theory, perturbations in the dystrophin-associated components lead to loss of membrane integrity. This is evidenced by increased permeability of muscle fibers to intravenously administered Evans blue dye as well as leakage of muscle-specific enzymes into the serum. This loss of membrane integrity eventually manifests itself as fiber degeneration. Thus, understanding the structural organization of the DGC is critical for understanding the function of this complex. Our findings represent the first account of SPN's localization in normal muscle, the expression of SPN in mutant mice, and the molecular associations of SPN within the DGC. We report that SPN, found at the sarcolemma of skeletal, cardiac, and diaphragm muscles, is also expressed at many specialized muscle membrane interfaces, including the NMJ and MTJ , as well as at muscle spindles (data not shown). SPN is also expressed in smooth muscle, where it is part of a unique smooth muscle SG–SPN complex (Straub, V., and K.P. Campbell, personal communication). Although SPN seems to be predominantly expressed in muscle, we detect SPN transcripts in many nonmuscle tissues . Consistent with this, our examination of dystrophin transgenic mice indicates that SPN may be associated with nonmuscle isoforms of dystrophin, such as Dp71 . Further experimentation is necessary to determine whether SPN protein is present in these nonmuscle tissues. The discovery that a subset of dystrophin-associated proteins (i.e., dystrophin, DG, and ε-SG) is present in a broad array of cell types is a provocative finding since all tissues are not subjected to the same shear stresses as muscle. This suggests that the DGC may serve a more fundamental role in the cell, in addition to the structural one ascribed to the DGC in muscle. We now show that SPN's localization to the sarcolemma is compromised in dystrophin and utrophin double null mice. SPN's enrichment at the NMJ is achieved by its association with utrophin. It has been suggested that upregulation of utrophin compensates for loss of dystrophin . Indeed, we have now demonstrated that the EOMs of mdx mice, which are spared from the pathological features of muscular dystrophy, express utrophin and SPN throughout the sarcolemma. If utrophin can functionally replace dystrophin, then it may be possible to upregulate utrophin expression in Duchenne muscular dystrophy patients . Our current data lend credence to the proposed theory that sarcolemma expression of utrophin would completely restore the dystrophin-associated proteins to the muscle plasma membrane. The DGC can be broken down into at least three interconnected subcomplexes: dystrophin, the DGs, and the SGs. Using several independent criteria, we demonstrate that SPN's localization to the sarcolemma is dependent on an intact SG subcomplex. SPN is completely absent from the sarcolemma, NMJ, and MTJ of the SG-deficient BIO 14.6 hamster and Sgca-null mouse. The preferential association of SPN with the SGs is demonstrated by biochemical isolation of the SG–SPN subcomplex. Alkaline treatment of purified DGC causes dissociation of the complex into distinct subcomplexes, where SPN preferentially associates with the SG containing fractions . Likewise, in the absence of dystrophin, the remaining extrajunctional dystrophin-associated proteins dissociate into distinct protein complexes, where SPN's specific interactions with the SGs are maintained . Furthermore, we reconstitute the SG–SPN complex in a recently developed heterologous cell system, which lacks muscle specific proteins . Previous work from our group has shown that mutations in an individual SG result in intracellular accumulation of the SG subcomplex . These experiments suggest that obligatory steps in the biosynthetic pathway for SG subcomplex assembly cannot occur if individual SG proteins are aberrant or missing . Taken together, our in vivo experiments now indicate that assembly of the SG subcomplex is a prerequisite for targeting and stabilization of SPN to the sarcolemma, as illustrated in Fig. 10 . We currently do not know the molecular basis of the interaction between the SG–SPN and DG subcomplexes. It is clear, however, that proper structural alignment of these two subcomplexes, along with dystrophin, is required for DGC function and prevention of muscular dystrophy. The data presented in the current study are also consistent with our finding that SG-deficient LGMD patients also lack SPN (Crosbie, R.H., and K.P. Campbell, personal communication). Although SPN is tightly associated with the SGs, SPN bears no structural homology to the SGs. To date, there are five known SGs, including the ubiquitously expressed ε-SG, which exhibits >40% amino acid identity to α-SG . ε-SG shares all the structural features of the skeletal muscle SGs, but is also expressed in many nonmuscle tissues. β-, γ-, and δ-SG are type II transmembrane proteins, while α- and ε-SG are type I membrane proteins with an NH 2 -terminal signal sequence. ε-SG expression is not perturbed by targeted deletion of the α-SG gene, suggesting that ε-SG is not an additional member of the α-, β-, γ-, δ-tetrameric SG subcomplex in skeletal muscle . Each of the SGs have a five cysteine residue motif in its extracellular domain, which is unique to this group of proteins. The SGs also possess one or more consensus sites for glycosylation and treatment with PNGase F has been shown to shift the molecular weight of these proteins. SPN, on the other hand, has many characteristics that distinguish it from the SGs. Most obviously, SPN is predicted to have multiple transmembrane domains and has no consensus sites for N-linked glycosylation. Consistent with this, treatment of purified DGC with PNGase F does not alter SPN's molecular weight (data not shown). Thus, SPN represents the first non-SG protein to be associated with the SG subcomplex of the DGC. The tight association of SPN with the SGs is consistent with SPN's homology to the tetraspan superfamily of proteins. The tetraspans are thought to function as facilitators of transmembrane protein interactions, and we suspect SPN serves to coordinate protein–protein interactions within the DGC. The results of our study provide support for this notion, since we find that SPN is intimately associated with at least one subcomplex of the DGC. Further examination of SPN's interaction with other DGC subcomplexes should provide significant insight into how the DGC is structurally organized, which is critical for understanding the function of this complex. | Study | biomedical | en | 0.999995 |
10189376 | D. discoideum wild-type strain AX2 and mutant strains were cultivated at 21°C, either on standard medium agar plates with Klebsiella aerogenes or axenically in liquid nutrient medium in shaking suspension at 150 rpm or submerged in plastic culture dishes. All reagents were purchased from Sigma Chemical Co. , if not stated otherwise. Antibodies against α- l -fucosidase (mAb 173-185-1) and coronin (mAb 176-3D-6) were kindly provided by Dr. G. Gerisch (Max-Planck-Institut for Biochemistry, Martinsried, Germany), and anti-vacuolin antibody (221-1-1) was provided by Dr. M. Maniak (MPI for Biochemistry, Martinsried, Germany). The hybridoma cell line ACTI that produces an mAb against D. discoideum actin was purchased from the American Type Culture Collection. Antibody against murine β-COP (mAb E5A3) was a gift from Dr. T. Kreis (Geneva, Switzerland). A cDNA library from HS2205 growth phase cells was provided by Dr. R. Gräf (A.-Butenandt-Institut für Zellbiologie, Munich, Germany). Standard techniques were used for cloning, transformation, and screening . The REMI of plasmid DNA was performed on the D. discoideum mutant strain pII/Ia2 that lacks both profilin isoforms . 10 μg of BamHI linearized plasmid pUCBsrΔBam along with 4 U DpnII were used for electroporation of 5 × 10 7 profilin-minus cells essentially as previously described . The transformants were selected with 4 μg/ml of blasticidin S (ICN Biochemicals Inc.) for 10 d, and cloned on K. aerogenes by spreader dilutions. About 4,000 primary transformants from several transformations were tested, and six independent colonies showing a rescued fruiting body formation were isolated; one of them (RB2) was chosen for further analysis. The integrated plasmid along with 2.6 kb of flanking genomic DNA was excised with ClaI, and the 7.2-kb DNA piece was cloned in Escherichia coli. An EcoRI fragment of 2.5 kb and shorter fragments of 2.0 and 1.1 kb generated by treatment with exonuclease III (Erase-a-Base; Stratagene) were subcloned in pUC19 . The fragments were sequenced with the chain termination dideoxy method using uni, reverse, and sequence specific primers. The isolated genomic sequence of the disrupted gene lmpA had a size of ∼1.2 kb, it contained the 3′ end of the coding region but lacked the 5′ end. Therefore, a λExCell cDNA library was screened as described previously . For screening, a 0.8-kb PCR fragment close to the known 5′ end of the genomic sequence was used and labeled with [α- 32 P]dATP with the Prime-It random primer kit (Stratagene). From one positive clone the cDNA insert was amplified by PCR using primers of the λExCell flanking regions, cloned into pUC19, and sequenced. The isolated cDNA had a size of ∼1.6 kb, contained the 5′ end, and overlapped with the 5′ region of the originally isolated ClaI fragment. To exclude possible errors resulting from PCR amplification of the λExCell cDNA clone, the sequence was confirmed with independently amplified and cloned PCR products. Using genomic DNA as a template, the 5′ region of the gene was amplified, cloned in pUC19 vector, and two independent clones were sequenced several times from both sides to obtain sequence information on the single intron. For the targeted homologous recombination the following was performed: the first 0.62-kb of the 5′ region of the lmpA gene starting with the ATG and including the intron, was amplified by PCR on genomic DNA; and inserted into the pUCBsrΔBam vector via XbaI and BamHI sites. The correct insertion of the genomic fragment was determined by DNA sequencing. Control transformations were carried out with the pUCBsrΔBam vector without insert. Transformations of profilin-minus cells with this vector were performed as described by Haugwitz et al. and selection was performed as stated above. For immunofluorescence studies, cells were allowed to attach to coverslips for 30 min in liquid nutrient medium, washed with Soerensen phosphate buffer, fixed with cold methanol (10 min), air dried, labeled and mounted as previously described . In case of α- l -fucosidase, cells were washed and starved for 6 h in Soerensen phosphate buffer before fixation. Secondary antibodies used for immunofluorescence included goat anti–mouse IgG and goat anti–rabbit IgG coupled to fluorescein or Cy3 (Dianova). The mounted cells were observed in an Axiophot microscope ( Carl Zeiss ). For counting nuclei, methanol-fixed cells were stained for 1 h with 0.5 μg/ml of 4,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co. ) in PBS. The cells were washed in DAPI-free buffer, rinsed in distilled water, and mounted. Standard immunofluorescence preparations were viewed on an inverted microscope (Leica DM IRBE; Leica GmbH) with an 100× objective. Images were acquired using the Leica TCS NT confocal imaging system, transferred to a personal computer , and further analyzed using the National Institutes of Health image public domain software and Adobe Photoshop 4.0. Axenically grown log-phase AX2 cells were harvested, washed in Soerensen buffer, resuspended in homogenization buffer (30 mM Tris/ HCl, 4 mM EGTA, 2 mM DTT, 30% sucrose, 5 mM benzamidine, 0.5 mM PMSF, 2 mM EDTA, 0.25% protease inhibitor cocktail, pH 8.0), and opened with a Parr bomb. The ruptured cells were further separated in cytosol and membrane fractions by centrifugation at 100,000 g for 1 h. By immunoblots, DdLIMP was found to be quantitatively localized in the pellet. The membranes were solubilized with 0.2% (final concentration) Triton X-100 for 60 min at 4°C. The material was subsequently centrifuged at 1,000 g for 5 min to remove particles and subjected to anion exchange chromatography on a DEAE column (DE52; Whatman Inc.) equilibrated with DEAE buffer (10 mM Tris/HCl, 1 mM EGTA, 1 mM DTT, 0.02% NaN 3 , 1 mM benzamidine, 0.5 mM PMSF, 0.2% Triton X-100, pH 8.0). Bound proteins were eluted with a linear salt gradient (0–400 mM NaCl in DEAE buffer). DdLIMP eluted at a conductivity between 4 and 8 mS/cm. Fractions containing DdLIMP were pooled, dialyzed against ConA buffer (20 mM Tris/HCl, 50 mM NaCl, 1 mM MnCl 2 , 1 mM CaCl 2 , pH 8.0) containing 0.1% Triton X-100, and loaded onto a 2.6 × 5 cm ConA–Sepharose column ( Pharmacia Biotech, Inc. ) equilibrated in ConA buffer. The column was washed extensively with ConA buffer without detergent, and bound proteins were eluted stepwise with 20, 100, and 500 mM of methyl-α- d -mannose in ConA buffer without detergent. If necessary, the DdLIMP containing fractions were further purified on gel filtration columns (Sephacryl S300; Pharmacia ), equilibrated in IEDANBP buffer (see below). We were able to obtain 1.1 mg of DdLIMP starting from 35 g of total membrane pellets (wet weight). The sedimentation assay was accomplished as described with minor modifications. Partially purified DdLIMP was incubated with IEDAN buffer (control) or with phospholipids for 30 min on ice in a total volume of 150 μl. After centrifugation at 100,000 g for 20 min at 4°C, 50 μl from the top of the supernatant was removed and stored on ice and the rest of the supernatant was discarded. The pellets were washed once with 150 μl buffer, centrifuged at 100,000 g for 10 min, and resuspended in 150 μl buffer. Supernatants and pellets were analyzed by SDS-PAGE followed by Coomassie blue staining or immunoblotting. All assays were carried out in the presence of 1 mM CaCl 2 and the PIP 2 was quantitatively sedimented under these conditions according to Flanagan et al. . DdLIMP-containing fractions were incubated 1:1 (vol/vol) with phospholipids or buffer (control) for 15 min on ice, centrifuged at 6,000 g for 10 min to remove aggregates, and 50 μl of the supernatant was immediately loaded on a Superose 6 PC 3.2/30 column (Smart System; Pharmacia Biotech, Inc. ) at 4°C. The column was equilibrated in IEDANBP buffer (10 mM imidazol, 1 mM EGTA, 1 mM DTT, 0.02% NaN 3 , 200 mM NaCl, 1 mM benzamidine, 0.5 mM PMSF, pH 7.6), and the flow rate was 40 μl/min. Fractions of 60 μl were collected and subjected to SDS-PAGE and Coomassie blue staining, or immunoblotting. Size calibration was carried out using ferritin (450 kD), catalase (240 kD), aldolase (158 kD), BSA (68 kD), and chymotrypsinogen (25 kD) as standard. To rule out a possible influence of Ca 2+ on the protein–phospholipid interaction, all gel filtration assays were repeated in the presence of 1 mM Ca 2+ . No significant variation was seen when duplicate experiments from both conditions were compared. Evaluation was done with the Smart Manager software package for OS/2. Preparation of DNA and RNA for Southern and Northern blot analyses was performed according to Noegel et al. . SDS-PAGE and immunoblotting followed standard procedures. Secondary antibodies used included: goat anti–mouse IgG and goat anti–rabbit IgG coupled to horseradish peroxidase (Dianova). The bound secondary antibodies were visualized with the enhanced chemiluminescence method (Nycomed Amersham ). Determination of protein concentration was done according to Lowry et al. with BSA as a standard. Rabbit actin was prepared from skeletal muscle according to Spudich and Watt . Recombinant and native Dictyostelium profilin were purified as described by Haugwitz et al. . Polyclonal antisera 3416 and 3417 against DdLIMP were raised by immunizing rabbits (Eurogentec) with a bacterially expressed polypeptide comprising the COOH-terminal half of DdLIMP, starting at amino acid 390. The recombinant protein carrying an NH 2 -terminal 6× His-tag was expressed in E. coli M15 cells using a pQE32 vector (Qiagen GmbH) and purified by Ni 2+ -NTA affinity chromatography (Qiagen GmbH). Compilation of DNA or protein sequences was done with the UWGCG program . Searches for similarities to other protein sequences were done with the BLAST program using the combined nonredundant entries of the Brookhaven Protein Data Bank, Swiss-Prot, PIR, and GenBank at the NCBI. Phylogenetic analysis was carried out with the program package PHYLYP . The REMI technique, first described by Schiestl and Petes , is a powerful method for tagging and cloning novel genes in model systems like D. discoideum , Candida albicans , Ustilago maydis , or in the vertebrate Xenopus . It has been used successfully to identify suppressors of developmental mutants in D. discoideum , and we applied it with the aim of isolating suppressor mutants for the profilin-minus phenotype. After 10 d of selective pressure the transformants were plated on K. aerogenes plates. Six colonies (RB1–RB6: REMI BamHI) were found to develop normally, whereas the development of the profilin-minus cells was always arrested at the finger stage. Southern analysis showed that RB1–RB4 and RB5–RB6, respectively, had the same restriction pattern. If one assumes that they share the same insertion and multiple independent insertions took place, then the screen would be near saturation, in accordance with earlier suppressor screens with the REMI method . One clone (RB2) was chosen for further analysis; the flanking regions of the other strains have not been determined. Determination of the number of nuclei showed that the prominent cytokinesis defect of the profilin-minus cells, resulting in the frequent appearance of giant multinucleated cells, was also suppressed in the RB2 mutant . However, the growth defects of profilin-minus cells were not restored. On bacterial lawn as well as in shaking culture at 120 rpm, RB2 had about the same growth rate as its parent strain pII/Ia2. Interestingly, the RB2 mutant seemed to be less prone to physical damage by shaking than the profilin-minus cells. Immunofluorescence studies showed that the broad F-actin rim frequently found in profilin-minus cells was not present in the RB2 strain. The F-actin distribution was indistinguishable from wild-type. Southern analysis of ClaI-digested genomic DNA from RB2, using a blasticidin S probe, showed a band of 7.2 kb in the REMI mutant. This band represents the plasmid used for mutagenesis with flanking sequences. The pUCBsrΔBam plasmid together with 2.6 kb of flanking genomic sequence were recovered from the RB2 strain and subjected to sequence analysis. Two open reading frames were found. One had a size of 1.176 kb and was disrupted by the pUCBsrΔBam vector at a DpnII restriction site; the second had a size of 0.6 kb and showed the opposite orientation. Both open reading frames were separated by a sequence whose A/T composition is typical for intergenic DNA in the Dictyostelium genome. Due to sequence homology with mammalian LIMP-II, the gene and the encoded polypeptide were named lmpA and DdLIMP, respectively. The coding region of lmpA lacked the ATG start codon but contained a TAA stop codon, the most frequently found termination codon in D. discoideum . Screening of a cDNA library resulted in the isolation of a cDNA clone with a size of ∼1.6 kb that lacked the 3′ region of the gene but contained the ATG start codon. The cDNA and the genomic sequence overlapped by ∼400 bp . With primers derived from the completed coding sequence, PCR was carried out on AX2 genomic DNA to confirm the results from the cDNA sequencing reactions. In addition, a single 98-bp intron was found near the 5′ end of the coding sequence. Examination of the deduced amino acid sequence of DdLIMP revealed a polypeptide with a length of 779 amino acids, a calculated molecular mass of 87.8 kD, and an isoelectric point of 4.4. A hydropathy profile according to the method of Kyte and Doolittle suggests two hydrophobic regions, one at the NH 2 terminus and one at the COOH terminus. The length of the apolar segment at the NH 2 terminus (24 amino acids) and the charge distribution in the flanking hydrophilic stretches suggest that this constitutes a signal anchor rather than a cleaved signal . In contrast, cleaved signal sequences have shorter apolar segments . In the case of Dictyostelium ponticulin, a protein reported to have a cleaved signal sequence, the hydrophobic segment contains only 13 residues . Rat LIMP-II, which is a mammalian homologue of DdLIMP was shown to contain an uncleaved signal peptide as well . There are 19 putative consensus sites for N -glycosylation present, and all of them are located in between the hydrophobic stretches . A comparison with the most recent databases using the BLASTP program revealed that DdLIMP was a novel D. discoideum protein related to LIMP-II of human and rat origin. Proteins of the LIMP-II class belong to the CD36, CLA-1, and LIMP-II gene family comprising single polypeptide membrane glycoproteins of apparently ubiquitous distribution in vertebrates and arthropods. As calculated with the program GAP (GCG program package), DdLIMP showed 35% similarity and 24% identity to rat LIMP-II, and 37% similarity and 25% identity to human CD36. There is a stretch of particular homology at the NH 2 -terminal side (between the NH 2 terminus and amino acid 160), followed by several insertions only found in DdLIMP, which renders this protein the largest member of the family thus far. A recently identified structural domain common to all known members of the CD36/LIMP-II family, the CLESH-1 motif , is located in this NH 2 -terminal region (amino acids 101–149 in DdLIMP, corresponding to amino acids 85–133 in CD36 from humans). Two out of three blocks of this motif are well conserved in DdLIMP. A second homologous stretch with several breaks spans the region between amino acid 640 and the COOH terminus. There is a tyrosine-based lysosomal sorting motif GY qa I at a consensus position in the COOH-terminal bona fide cytosolic tail. Interestingly, this motif is only found in vertebrate proteins of the LAMP 1 family , that show otherwise no significant sequence homology to DdLIMP. Mammalian LIMP-II lacks any tyrosine residues in this region and has a di-leucine sorting motif . Protein sequences of typical members of the CD36/ LIMP-II/CLA-1 family from different species were aligned and used for a phylogenetic comparison using the program PHYLYP . DdLIMP is located at the bottom of the tree corresponding to the assumed evolutionary origin of Dictyostelium . A BLASTP search with DdLIMP as query in the completed S. cerevisiae genome database (Stanford, CA) yielded no significant homologous sequences, making DdLIMP the most divergent member of this family. Southern blot analysis of AX2 genomic DNA digested with various restriction enzymes indicated that lmpA is a single gene in the Dictyostelium genome, and no obvious cross-reacting bands were detectable under conditions of high stringency. To test for the existence of related sequences, the Southern blots were reprobed at low stringency (30% formamide). Only in one out of seven restriction digests that were tested, was an additional, lower size band observed. By Northern hybridization analysis a transcript of 3.5 kb could be detected , which is in agreement with the size of the complete lmpA coding sequence with an additional poly-A tail. However, a smaller additional transcript of 1.4 kb was observed in some cases. It is not clear whether the smaller transcript occurs because of a cross-reaction of the probe or arises from posttranscriptional processing. Interestingly, for both of the mammalian lmpA homologues CD36 and LIMP-II, two or more transcripts have been demonstrated . The lmpA transcript is present at about equal amounts during all stages of the Dictyostelium development (not shown). The lmpA mRNA is not altered in the profilin-minus cells as compared to wild-type RNA (not shown). Northern analysis of the REMI mutant RB2 revealed a complete absence of the 3.5-kb transcript. However, a larger faint band of ∼8 kb hybridized with the full-length lmpA probe (not shown). This large transcript is most probably generated at the endogenous lmpA start of transcription and contains the integrated REMI vector. A polyclonal antiserum was raised by immunizing rabbits with the recombinant COOH-terminal half of DdLIMP. It recognized a single band of ∼120 kD in Western blots of AX2 homogenates, and after centrifugation at 100,000 g the signal was almost exclusively found in the membranous pellet . From quantitative immunoblots, one can estimate that DdLIMP has a cellular molarity of 0.5 μM, and constitutes ∼0.1% of total membrane protein. The band was unchanged under nonreducing conditions. The size of the band exceeds the calculated molecular mass of 87.8 kD, suggesting a posttranslational modification. DdLIMP bound to the lectin ConA and could be eluted with α- d -mannose, which also points towards modification by N -glycosylation. The same band was observed in the profilin-minus strain, but was clearly absent from the REMI mutant RB2. Truncated forms of DdLIMP were not detected in the RB2 strain by the polyclonal antiserum. DdLIMP could not be extracted from the membrane pellet with buffer containing 0.5 M NaCl, but was solubilized with Triton X-100 (not shown), a typical behavior of integral membrane proteins. Taken together, these results suggest the following model: DdLIMP is an integral membrane protein, both the NH 2 and the COOH terminus are cytosolic, whereas the major part of the protein in between the two membrane spanning regions is presumably highly N -glycosylated and intravesicular. Similar hairpin-like structures have also been proposed for CD36 and LIMP-II . Immunofluorescence studies on the distribution of DdLIMP in AX2 cells showed that the majority of the protein localized to small vesicles (<0.5 μm) of varying size . A particular membrane staining was not observed. However, it cannot be excluded from our data that part of the signal was located at the plasma membrane. This would not be surprising in the light of membrane recycling events occurring from postlysosomes. The same localization could be observed in the profilin-minus cells (not shown), whereas the DdLIMP staining was completely absent in the REMI mutant RB2 . DdLIMP was not only found in vesicles, but it also localized to punctated rings surrounding larger vesicular structures (diam: 2.1 ± 0.6 μm, n = 21). These larger fluorescent rings corresponded to phase-opaque vesicles . Double staining with an mAb against the lysosomal enzyme α- l -fucosidase was carried out using confocal microscopy (not shown). This protein, found in a major population of lysosomes, is developmentally regulated. Therefore, colocalization studies were conducted with cells after 6 h of starvation. In control experiments, it was shown that the DdLIMP localization pattern was not changed during this developmental stage. Surprisingly, it was found that there was no colocalization of both proteins. To further investigate the nature of the DdLIMP-positive structures, double staining experiments with an heterologous antibody against the COPI coatomer protein, β-COP, were carried out. It was found that there was a substantial colocalization in the punctated rings, but not in the smaller vesicles . β-COP plays a role in the early stages of the secretory pathway, as well as in early to late endosomal transfer . Double-labeling experiments with coronin, a protein found at phagocytically active cell projections known as crowns, revealed only minor colocalization . However, in some cases, a double labeling of ringlike structures was observed. Essentially, the same was observed in double labeling experiments with vacuolin, a marker for postlysosomes ; only a small subpopulation of the punctated rings was both vacuolin- and DdLIMP-positive. These findings indicate a presence of DdLIMP in the endosomal pathway. In fact, some of the large punctate rings may be macropinosomes. DdLIMP was partially purified from D. discoideum AX2 cells by conventional chromatography and used for in vitro binding assays. It was first tested whether DdLIMP was able to directly interact with profilin. This was done by immunoprecipitation of cell homogenates with mAbs for profilin I and II , or anti-DdLIMP polyclonal antiserum, and subsequent immunostaining. No coprecipitation of DdLIMP and either of the profilins was observed; the same result was obtained when profilin was precipitated by poly( l -proline) coupled to agarose beads (not shown). Recombinant profilin I and II were incubated with DdLIMP or rabbit actin, treated with the chemical cross-linking agent EDC as described by Haugwitz et al. , and analyzed by SDS-PAGE and immunoblotting. The control clearly showed cross-linking of both profilins to actin, whereas no profilin–DdLIMP complexes were observed in the treatment group (not shown). Since actin is known to play a role in vesicle transport along the endolysosomal pathway in Dictyostelium , one might assume an interaction of the membrane protein DdLIMP with the microfilament system. To test this possibility, DdLIMP was incubated with rabbit actin under polymerizing conditions on ice for 1 h and the polymerized actin filaments were pelleted by centrifugation at 100,000 g for 30 min. No DdLIMP was detected in the pellets by analysis with SDS-PAGE and immunoblotting (not shown). To test the interaction with phospholipids, we incubated partially purified DdLIMP with PIP 2 in micellar form and centrifuged the mixture at 100,000 g. DdLIMP alone was found to be soluble, and was sedimented only in the presence of PIP 2 , whereas the contaminating peptide p60 stayed in the supernatant. To exclude that the interaction was only due to nonspecific interaction of the transmembrane domains of DdLIMP with the hydrophobic part of the phospholipid, control experiments with cationic phosphatidylcholine (PC) were carried out , and no interaction was found. Phosphatidylserine (PS), like PIP 2 , belongs to the class of anionic phospholipids, and has been described as a ligand for mammalian CD36 . Liposomes solely composed of negatively charged PS were able to sediment more DdLIMP than PC liposomes. However, there was still significantly less binding as compared to PIP 2 . DdLIMP also bound PIP 2 in mixed liposomes in the presence of a fivefold excess of PC . Under these conditions, a significant amount (∼50%) of DdLIMP was sedimented by the mixed liposomes. About the same affinity was found for PC/PI(4)P mixtures, but significantly less sedimentation was observed with PC/PS liposomes . Since the major part of the DdLIMP protein is proposed to localize to the lumen of endolysosomal compartments of putative low pH, the pH dependence of the protein–phospholipid interaction was also tested. It was found that the binding characteristics, at pH 5.0, were comparable to the assays done at pH 7.6. In contrast, binding was abolished at pH 9.0. In gel filtration assays, DdLIMP bound to PIP 2 micelles but not to control liposomes. A Superose 6 column was used with the Smart System that allowed the application of very small samples and had excellent detection capabilities. Calibration of the column with molecular mass standards indicated a high reproducibility of the retention volume. When DdLIMP was applied to the column in the absence of phospholipid, it eluted at 1.51 ± 0.02 ml (mean ± SD for three experiments), which corresponds to a molecular mass of 240 kD . The contaminating p60 eluted at 1.74 ml , corresponding to 50 kD. The discrepancy in the observed elution of DdLIMP (240 kD) and the relative mass calculated from the electrophoretic behavior in reducing gels (120 kD) might reflect a dimerization of DdLIMP, which has been observed for the higher eukaryote homologues SR-BI , CD36 , and FAT , but could also arise from unusual elution behavior and the presence of tightly bound detergent molecules. However, the elution behavior did not change when the protein preparation was incubated with PC or PS (not shown). Upon addition of PIP 2 micelles, which are reported to have a relative molecular mass of 93 kD (aggregation number 82) in aqueous solutions , the elution of DdLIMP shifted to 1.42 ml (440 kD), whereas the p60 peak was essentially unchanged at 1.72 ml (60 kD). The same was observed with a phospholipid mixture from biological origin (brain phospholipids, Folch fraction), which contains 50% PS and 10% inositol phospholipids. To make sure that the observed rescue phenotype of the REMI mutant RB2 was really due to the disruption of the lmpA gene, homologous recombination was carried out on the profilin-minus strain. The gene disruption vector included a 620-bp genomic DNA of the lmpA gene together with a blasticidin S resistance cassette. The transformants were selected for 10 d, cloned, and screened for a wild-type–like phenotype. In control transformations with the vector alone, no suppressor colonies were observed. One clone (T1.5) with phenotypically normal fruiting bodies and wild-type–like cytokinesis in shaking culture was chosen for further analysis. Immunofluorescence studies showed a decrease in the DdLIMP signal and densitometric scanning of immunoblots revealed that DdLIMP was still present, but reduced by a factor of two . About 50 mutants that were unable to form fruiting bodies were picked randomly and analyzed as nonsuppressor controls. None of them had an altered DdLIMP signal, as judged by immunoblotting. Southern hybridization assays with genomic DNA from the T1.5 clone confirmed a disruption of the lmpA gene . A radiolabeled probe specific for the resistance cassette detected only one band , indicating a single integration of the vector in the genome. The integration of the bsr vector restored the complete lmpA open reading frame, but apparently displaced the endogenous ATG and the 5′ untranslated sequence , resulting in the appearance of a second larger transcript . The partial reduction of the DdLIMP concentration resulted in a very similar, but not identical phenotype as compared to the original REMI clone RB2. The gene disruption mutant had a reduced cell size (∼20% smaller) as compared to RB2. Even though both suppressor strains were able to form phenotypically normal fruiting bodies, they were not able to produce viable, detergent-resistant spores. This was tested by treatment of fully developed aggregates with 0.5% Triton X-100 and subsequent plating on bacteria. Wild-type spores easily withstood that treatment, whereas in the suppressor strains no colonies were observed. Sorocarps from both suppressor strains were completely devoid of wild-type– like oval encapsulated spores as judged by light microscopy. To gain further evidence on the role of DdLIMP, a lmpA -minus strain was created by homologous recombination in the wild-type strain AX2. For this purpose, the gene disruption vector containing a 620-bp homologous fragment, from the 5′ end of the lmpA gene (described above), was slightly modified to prevent restoration of the complete open reading frame by recombination of endogenous and vector lmpA sequence. The vector was digested with the restriction enzyme AccI (that cuts once in the lmpA coding sequence), treated with S1-nuclease, and religated. By this treatment, a 2-bp deletion was introduced in the lmpA open reading frame, thus giving rise to a frameshift mutation. AX2 was transformed with the ΔAccI vector and the transformants were selected for 10 d with blasticidin S. 600 clones were picked randomly and analyzed by immunofluorescence and immunoblotting for the absence of the DdLIMP signal. Only one clone (T2.25) was found to have an obviously reduced amount of DdLIMP in immunoblots and a very weak staining in immunofluorescence, pointing towards a homologous recombination event. Southern analysis of genomic DNA revealed a shift in the lmpA bands for T2.25. In the case of strain T1.17, a nonhomologous control transformant, the endogenous lmpA bands were unchanged and additional bands occurred that corresponded to the vector. PCR analysis on genomic DNA using vector and gene specific primers further confirmed a homologous integration for T2.25 and a nonspecific integration for T1.17. Northern analysis with a full-length lmpA probe revealed a transcript indistinguishable from wild-type in T1.17. In the case of T2.25, the 3.5-kb mRNA was reduced twofold and a second, larger transcript had appeared, comparable to the full-circle clone T1.5 . The gene disruption strain T2.25 showed normal cytokinesis and developed into wild-type–like fruiting bodies. However, the number and size of fruiting bodies was reduced as compared to the wild-type. The sorocarps contained viable, detergent-resistant spores, but their number was strongly decreased. In comparison to the wild-type strain, AX2, the number of round uncoated spores in the sorocarp was increased by a factor of five (as judged by light microscopy). The T2.25 strain had normal growth rates on bacteria, but showed greatly reduced growth in submersion culture. Furthermore, it was unable to withstand shearing forces in shaking culture at 150 rpm . After 2 d in shaking culture, many small vesicle-like fragments appeared that might be due to extensive membrane shedding. The control strain T1.17 grew normally, although slightly slower than the wild-type under all conditions tested, and was not impaired in growth at 150 rpm. Similar to the full-circle clone T1.5, the cell size of T2.25 was reduced slightly, whereas the control strain T1.17 had a size distribution comparable to the wild-type. We have used the REMI approach to investigate regulatory cascades upstream of profilin by screening for suppressor mutants in profilin-minus D. discoideum cells. The disrupted gene lmpA was recovered from the mutant and coded for an integral membrane glycoprotein with PIP 2 binding activity, constituting the first reported Dictyostelium homologue of the CD36/LIMP-II/CLA-1 gene family. Targeted homologous recombination of lmpA in a profilin-minus background resulted in a similar suppressor phenotype. It has been shown that there are many high molecular weight, low pI, integral membrane glycoproteins in purified lysosomal membranes from D. discoideum . However, no LAMPs or LIMPs have been reported to date. DdLIMP showed significant homology to the CD36/LIMP-II/CLA-1 family of integral membrane proteins from mammalian or insect origin. The overall structural features appear to be quite well conserved evolutionarily, from the position of the two transmembrane domains to the glycosylation pattern. Apart from its function in cell adhesion and signal transduction , CD36 and other members of the gene family, notably the scavenger receptor SR-BI and the CD36 homologue FAT from adipocytes , have been identified as lipid receptors that bind long-chain fatty acids , PI, and PS . The adipose CD36 homologue FAT has been reported to exist as a homodimer and, possibly, as part of an oligomeric transport complex . Since DdLIMP was localized on vesicles rather than at the plasma membrane, it bears more similarity to LIMP-II than to the other members of the CD36/LIMP-II/CLA-1 family shown to be cell surface molecules . However, our immunofluorescence studies identified the DdLIMP-positive vesicles and the α- l -fucosidase–positive lysosomes as two different populations. According to Souza et al. , there are two separate populations of lysosomes in D. discoideum , which are either acidic and glycosidase-rich or of more neutral pH and rich in cysteine proteases. Thus, DdLIMP may be localized in the cysteine protease-containing lysosome population. DdLIMP was also found in larger punctate ringlike structures (diam 2 ± 0.6 μm) that resemble macropinosomes or postlysosomes that are reported to be less acidic than lysosomes . An intriguing feature of DdLIMP is its apparent divergent lysosomal sorting motif. Mammalian LIMP-II shows a typical di-leucine type motif (Leu-Ile) in the short COOH-terminal tail that extends into the cytosol , whereas the corresponding sequence in DdLIMP ( GY QA I ) more closely resembles the tyrosine-based signal of the LAMP1 type . Mammalian LIMP-II interacts with the recently identified nonclathrin AP-3 adaptor complex via its di-leucine signal . On the other hand, the proteins of the LAMP1 family have only one membrane-spanning domain and the sequences are unrelated to DdLIMP. The tyrosine motif is thought to act as a binding site for adaptor protein complexes (AP1 or AP2), and it has been shown to target LAMP1 to lysosomes , or plasma membrane proteins to endosomes . Even though PI lipids constitute only a small fraction of total lipids in the membranes of eukaryotic cells, they are thought to play a pivotal role in vesicle transport, signal transduction, and cytoskeletal regulation. PIP 2 constitutes a central component in the polyphosphatidylinositide pathway by serving as a precursor to several inositol lipid second messengers and directly regulating protein localization and activity of many actin-binding proteins. In Dictyostelium endolysosomes, PIP 2 accounts for 9% of total lipids, as compared to 11% in the plasma membrane . Most of the many proteins described that bind to PIP 2 are cytosolic. Thus far, only two integral membrane proteins with PIP 2 binding activity have been described: the inward rectifier potassium channel from cardiac muscle and the human intercellular cell adhesion molecules-1 and -2 (ICAM-1 and -2) . None of the classical PIP 2 -binding sequence motifs can be found in DdLIMP. There is considerable heterogeneity in the primary sequence domains and structures that are reported to bind to inositol phospholipids . However, in all cases the interactions are mediated by positively charged amino acid residues on the protein and the negatively charged phosphate groups on the inositol headgroup. In addition to the electrostatic interactions, hydrophobic segments are thought to contribute to binding . Given the assumed membrane topology of DdLIMP and the localization of inositol phospholipids in the cytoplasmic leaflet of cellular membranes , it is feasible that the short cytosolic tails of DdLIMP account for binding to the negatively charged inositol headgroup. ICAM-1, the first described cell adhesion molecule with PIP 2 -binding activity, is an otherwise unrelated protein that has a relatively short (27 amino acid) COOH-terminal tail containing a high amount of positively charged residues. This tail is sufficient for phospholipid binding and induces interaction of ICAM-1 and ezrin . Comparison of DdLIMP to ICAM-1 and ICAM-2 (both binding to PIP 2 ) shows a considerable degree of similarity in this region , whereas ICAM-3, which does not bind to PIP 2 , shares no significant similarities with DdLIMP. Since disruption of the lmpA gene suppresses the profilin-minus phenotype there must be some interaction, direct or circumstantial, between the cytosolic actin-binding protein, profilin, and the integral membrane protein, DdLIMP. A direct binding of profilin to DdLIMP is unlikely, as judged from our own in vitro binding studies and the lack of polyproline stretches in DdLIMP, the most characteristic feature of recently found profilin-binding proteins like VASP , diaphanous from Drosophila , and p140mDia from mammals . In the fission yeast S. pombe , mutations in the essential gene sop2 or in arp3 were reported to rescue the temperature-sensitive lethality of a profilin mutant . The gene product Sop2p shows homology to the β-transducin family and is thought to interact with a protein complex containing profilin, actin, and Arp3p. In S. cerevisiae , an interaction of profilin with the vesicular transport system responsible for exocytosis was found by identification of SEC3, a component of the exocyst protein complex, as a profilin synthetic lethal gene . None of those bona fide profilin ligands or interacting partners bear similarity to the membrane proteins of the LIMP-II class. Interestingly, unpublished observations (Cardelli, J., and M. Schleicher) showed that the profilin-minus mutant exhibited a strong defect in endocytosis, exocytosis, and the secretion of the lysosomal enzyme acid phosphatase. The vesicle transport defects are partially restored in both the original REMI mutant RB2 as well as the recapitulation strain T1.5. There are several lines of evidence supporting a connection between the signal transduction pathway, known as PI cycle, profilin, and actin-dependent vesicle transport during endocytosis or exocytosis. Knocking out the PI3-kinases in macrophages inhibited the completion of macropinocytosis and phagocytosis . Accordingly, the loss of the related kinases DdPIK1 and DdPIK2 in Dictyostelium amebas had severe defects in endocytosis, transport from lysosomes to postlysosomes, and the actin cytoskeleton . Recently, human profilin has been shown to bind the lipid products of PI3-kinases with higher affinity than PtdIns(4,5)P 2 . Apart from being a possible downstream target of the PI3-kinases in the endosomal pathway, there is a more direct evidence for an interaction of profilin with phagocytosis in Dictyostelium. It has been shown that actin and several actin-binding proteins are associated with phagocytic cups and early phagosomes , and the same holds true for profilin (our unpublished observations). The uptake of fluid and particles by D. discoideum depended on the actin cytoskeleton . Also, it was shown recently that depolymerization of F-actin by cytochalasin A inhibited exocytosis . The fact that myosin I mutants exhibit defects in endocytosis, exocytosis, and secretion of lysosomal enzymes further underlines the tight linkage of the actin cytoskeleton to lysosome-related membrane events in Dictyostelium . Profilin, like other cytoskeletal proteins , binds with high affinity to the membrane phospholipid PIP 2 and to a lesser extent to its precursor PIP . The phosphoinositide metabolism is proposed to be responsible for the partitioning of profilin between the plasma membrane and the cytosol , which is thought to be crucial for the regulation of the microfilament system. It has been proposed that a substantial fraction of total PIP 2 in the inner leaflet of the plasma membrane is bound by profilin . Binding to profilin inhibits the hydrolysis of PIP 2 by phospholipase Cγ, and plays an important role in the polyphosphoinositide pathway, which has been reported to involve Rac-driven GTPase activity . The loss of profilin in ameba cells results in a severe and multiple phenotype that might be mediated not only by the increased amount of F-actin, but also by an altered spectrum of phospholipids. This in turn acts on polyphosphoinositide-regulated actin-binding proteins. In the profilin-minus mutants, DdLIMP as a putative receptor or transporter of phosphatidylinositides would not be counterbalanced by the abundant PIP 2 -binding protein profilin. A disruption of the gene that codes for the lipid carrier could compensate the loss of profilin and rescue most of the phenotypic changes. | Study | biomedical | en | 0.999996 |
10189377 | Postmortem articular cartilage was aseptically removed from macroscopically normal femoral condyles and tibial plateaux of human knee joints. Donors had died from a variety of diseases unrelated to the locomotor system and were undergoing routine hospital autopsy. Cartilage was sampled from 8 males (mean age, 68 yr; range 58–83 yr) and 17 females (mean age, 76 yr; range 37–93 yr). Cartilage from different anatomical regions of the knee joint were pooled and chondrocytes were isolated by sequential enzyme digestion at 37°C in 95% air/5% CO 2 with 0.25% trypsin ( GIBCO BRL ) for 30 min and 3 mg/ml collagenase (type H; Sigma Chemical Co. ) for up to 24 h as described previously . Cells were seeded in Ham's F12 medium supplemented with 10% FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin to a final density of 5 × 10 5 /ml in 55-mm plastic petri dishes (Nunc), and cultured in a 95% air/5% CO 2 incubator at 37°C. Primary, nonconfluent, 5–10 d cultures of chondrocytes were used in all experiments in an attempt to limit changes in gene expression (dedifferentiation). Morphologically, the cells studied were typically flattened with a polygonal cell shape and did not show the fibroblastic appearance of dedifferentiated chondrocytes . Immunological and molecular analyses confirmed production of similar ECM molecules (type II and VI collagen, fibronectin, and keratin sulphate) and expression of identical integrin profile subunits (β1, β5αV, α1, α3, and α5) to that of HAC in vivo and after initial cell extraction . In our experience these chondrocytes show a consistent and reproducible membrane hyperpolarization response to 0.33 Hz mechanical stimulation . We have assessed the electrophysiological response of chondrocytes from knee joint articular cartilage of >80 different individuals and observed no significant difference in the membrane response in cells with respect to gender, age, and cause of death as it relates to patients without a history of locomotor system involvement (unpublished observations). To investigate whether the membrane hyperpolarization response was critically dependent on IL-4, chondrocytes from the joints of mice heterozygous for IL-4 or IL-4–deficient were studied. Chondrocytes were isolated by sequential enzymatic digestion and cultured as described above. The IL-4 knockout mice used have been previously described . Mice were obtained from a colony maintained by Dr. M. Norval (Department of Medical Microbiology, Edinburgh University, Edinburgh, United Kingdom) with permission for use of these animals provided by Professor Horst Bluethmann (Hoffmann-LaRoche AG, Basel, Switzerland). Total RNA was extracted from cultured chondrocytes as described in the micro RNA isolation kit (Stratagene), using a denaturing buffer of 4 M guanidine thiocyanate, 0.75 M sodium citrate, 10% (wt/vol) lauroyl sarcosine, and 7.2 μl/ml β-mercaptoethanol. The quantity of RNA isolated was determined by spectrophotometry using the absorbance reading at 260 nm. Before cDNA synthesis, all RNA samples were incubated with DNase I (Life Technologies) for 15 min in the presence of RNase inhibitor (Life Technologies). Template cDNA was synthesized using 1–5 μg RNA, superscript II, and oligo dT(12–18; Life Technologies) according to the manufacturer's instructions. Primers specific for IL-4 , IL-4 receptor α , the common gamma chain , and IL-13 receptor α were used for the PCR reactions: IL-4 5′-TTTGAACAGCCTCACAGAGC-3′, 5′-TCCTTCACAGGACAGGAATT-3′; IL-4Rα 5′-CTTGTTCACCTTTGGACTGG-3′, 5′-CTTGAGCTCTGAGCATTGCC-3′; γc 5′-CTCCTTGCCTAGTGTGGATGG-3′, 5′-CACTGTAGTCTGGCTGCAGAC-3′; and IL-13Rα 5′-GTGAAACATGGAAGACCATC-3′, 5′-GTGAAATAACTGGATCTGATAGGC-3′. A typical 20-μl PCR reaction contained 16 mM ammonium sulphate, 67 mM Tris/HCl, pH 8.8, 0.01% (vol/vol) Tween 20, 1 μM of each primer, 2 μl cDNA, 100 μM dNTPs, 0.1% (wt/vol) BSA, and 0.25 U Taq polymerase (Bioline). The magnesium chloride concentrations for each primer pair were: IL-4, 4 mM; IL-4Rα, 2.5 mM; γc, 2 mM; and IL-13Rα, 1.5 mM. The following program was used for all reactions: 94°C for 3 min; 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min 30 s; 72°C for 10 min. PCR products were analyzed by electrophoresis using a 1% (wt/vol) agarose gel. PCR products were cloned into the TA cloning vector (Invitrogen Corp.) as described in the manufacturer's protocol. Each insert was sequenced using the Sanger dideoxy chain termination method , modified according to the protocol provided with the sequenase kit (United States Biochemical Corp.). The technique and apparatus used have been previously described in detail . For the induction of pressure-induced strain (PIS), 55-mm diameter plastic petri dishes (Nunc) were placed in a sealed pressure chamber with inlet and outlet ports. The chamber was pressurized using nitrogen gas from a cylinder, at a frequency determined by an electronic timer controlling the inlet and outlet valves. The standard stimulation regimen used was a frequency of 0.33 Hz (2 s on/1 s off) for 20 min, 37°C, at a pressure of 16 kPa above atmospheric pressure. This system was shown to produce microstrain on the base of the culture dish . Membrane potentials of cells were recorded using a single electrode bridge circuit and calibrator, as previously described . Microelectrodes with tip resistances of 40–60 MΩ and tip potentials of ∼3 mV were used to impale the cells. Membrane potentials of isolated cells were measured and results were accepted if, on cell impalement, there was a rapid change in voltage to the membrane potential level that remained constant for at least 60 s. Experiments were performed at 37°C. The membrane potentials of 5–10 cells were measured before and after the period of PIS. Anticytokine, antiintegrin, and anticytokine receptor antibodies were added to chondrocytes 30 min before mechanical stimulation. Membrane potentials were measured before and after addition of antibody and after the period of mechanical stimulation. Antibodies had no effect on the resting membrane potential. Antibodies remained in contact with cells during cyclical PIS and when poststimulated membrane potentials were measured. Antibodies against IL-1β, IL-4, IL-4Rα, and γc were obtained from R&D Systems, Inc. Anti–β1 integrin (P4C10) and anti–αVβ5 integrin (P1F6) were obtained from Life Technologies. For each condition tested, at least three experiments were performed on different cells from different donor knees on different days. Membrane potential of chondrocytes was measured before and 10 min after the addition of recombinant IL-1β, IL-4, TGF-β1, and interferon gamma (IFN-γ; R&D Systems). To investigate signaling molecules involved in IL-4–induced hyperpolarization chondrocytes were treated, in separate experiments, with a number of pharmacological inhibitors of cell signaling for 30 min before addition of recombinant IL-4. The reagents used ( Sigma Chemical Co. ) were: neomycin, an inhibitor of PLC ; flunarizine, an inhibitor of IP3-mediated release of Ca 2+ from the ER ; genistein, a tyrosine kinase inhibitor ; apamin, a specific blocker of SK channels ; and gadolinium, a blocker of stretch-activated ion channels . The mean, SD, and standard error of the mean were determined in each experiment. For statistical comparisons, when the F ratio of the two variances reached significance, the nonparametric Mann-Whitney test was used. When the ratio did not reach significance, the Student's t test was used. HAC subjected to PIS at 0.33 Hz, 37°C for 20 min undergo hyperpolarization of the plasma membrane by ∼45% (Table I ). Conditioned medium from mechanically stimulated cells, when added to unstimulated chondrocytes, caused membrane hyperpolarization of these cells similar to that of the directly mechanical strained chondrocytes (Table I ), demonstrating the presence of a soluble, transferable factor secreted by the mechanically stimulated chondrocytes. 1 μg/ml P4C10, an anti–β1 integrin antibody, when incubated with chondrocytes for 30 min at 37°C before stimulation, inhibited the hyperpolarization response to mechanical stimulation. Medium from cells mechanically stimulated in the presence 1 μg/ml P4C10, when transferred to unstimulated cells, did not significantly alter the membrane potential of these cells (Table I ). In contrast, 1 μg/ml P1F6, an anti–αVβ5 integrin, had no effect on 0.33-Hz cyclical microstrain-induced hyperpolarization or production of a transferable factor that could induce membrane hyperpolarization of unstimulated chondrocytes. When monolayer cultures of HAC were incubated in separate experiments with a panel of recombinant human cytokines (IL-1β, IFN-γ, TGF-β1, and IL-4), known to be involved in the regulation of chondrocyte metabolism and potentially could function as autocrine/paracrine signaling molecules, a change in membrane potential was seen . Addition of IL-4 resulted in membrane hyperpolarization, whereas the other cytokines induced membrane depolarization. The effect of IL-4 on the membrane potential of human chondrocytes was dose-dependent over a range between 100 fg/ml and 10 ng/ml. A 17% hyperpolarization response was elicited at concentrations as low as 10 fg/ml and a maximal response was obtained with 5–10 pg/ml (results not shown). Using immunohistochemical techniques we have shown IL-4 to be present in HAC . However, its production by these cells and the expression of IL-4 receptors were not previously described. RT-PCR on total RNA isolated from primary cultured chondrocytes using primers specific for IL-4 resulted in amplification of a 269-bp region of DNA . This DNA region, when cloned and sequenced, displayed 100% identity to the published sequence of human lymphocyte IL-4 mRNA . RT-PCR reactions using primers to IL-4Rα, γc, and IL-13Rα revealed DNA products of 465, 356, and 450 bp, respectively , corresponding to the components of both the type I IL-4 receptor (IL-4Rα/γc) and type II receptor (IL-4Rα/IL-13Rα). Neutralizing antibodies to IL-4 abolished the hyperpolarization response to cyclical strain, whereas neutralizing antibodies to IL-1β had no effect . Specific antibodies to IL-4Rα (10 μg/ml) prevented the hyperpolarization response of chondrocytes to mechanical stimulation, whereas inhibitory antibodies to the γc subunit had no effect on the response . Anti–IL-4 antibodies (1 μg/ml), added to medium after mechanical stimulation but before transfer of that medium to unstimulated cells, prevented subsequent hyperpolarization of unstained cells (Table II ). Chondrocytes isolated from the articular cartilage of knee joints from IL-4 knockout mice did not show a significant change in membrane potential after 20 min of mechanical stimulation at 0.33 Hz . In contrast, chondrocytes isolated from knee joints of heterozygous mice showed a similar hyperpolarization response to mechanical stimulation as that seen with HAC. The hyperpolarization response of HAC to recombinant human IL-4 (10 pg/ml) was unaffected by P4C10 (anti–β1 integrin), genistein (a tyrosine kinase inhibitor), and gadolinium (a blocker of mechanosensitive ion channels; Table III ), although these agents were shown previously to inhibit the hyperpolarization response of HAC to mechanical stimulation . Neomycin (an inhibitor of PLC), flunarizine (an inhibitor of IP3-mediated release of Ca 2+ from the ER), and apamin (an SK channel blocker) each inhibited the chondrocyte hyperpolarization response to IL-4 (Table III ). This study has shown for the first time that IL-4 and its receptor are expressed by HAC. Furthermore this study also has shown that the cytokine receptor pair are involved in the integrin-dependent signaling pathway activated by 0.33-Hz cyclical strain that leads to the opening of SK channels and membrane hyperpolarization. Close associations between integrin and growth factor– mediated signaling in regulation of cell function are being identified. Cell adhesion–dependent activation of the Ras/ MAPK pathway may involve tyrosine phosphorylation of PDGF receptors . Angiogenic effects of a number of growth factors including basic fibroblast growth factor and vascular endothelial growth factor are integrin-regulated . Integrin-mediated cell adherence also has been shown to be important in cytokine gene expression in synovial fluid cells from patients with rheumatoid arthritis and by mast cells after Ig E receptor aggregation . Wilson et al. have demonstrated previously that mechanical strain induces growth of vascular smooth muscle cells via an autocrine action of PDGF. However, the growth-promoting effect required 36–48 h of mechanical stimulation and was associated with increased levels of PDGF mRNA, suggesting slow production and release of the cytokine rather than the rapid release of a preformed mediator after mechanical stimulation, as demonstrated in our system. It is unclear how integrin-mediated signaling causes IL-4 release. Rapid release of neurotransmitter from frog muscle motor nerve terminals after stretch is integrin-dependent and requires both intra and extracellular calcium . The data from our studies suggest that mechanical stimulation induced release of IL-4 by human chondrocytes after recognition and transduction of the mechanical signal by α5β1 integrin. Furthermore, activation of a signaling pathway involving tyrosine kinases, stretch-activated ion channels, and the actin cytoskeleton is consistent with other models of integrin-mediated mechanotransduction . IL-4 in turn binds to the chondrocyte IL-4 receptor heterodimer, IL-4Rα/IL-13Rα, initiating a signal cascade involving PLC and IP3-mediated Ca 2+ release and subsequent activation of SK channels, leading to K + efflux and membrane hyperpolarization. Coordinated activations of integrin and IL-4–associated signaling pathways in chondrocytes are of potential importance in regulating the structure and function of normal and diseased articular cartilage. Regulation occurs by mediating other biochemical responses to mechanical strain, e.g., proteoglycan synthesis , or altering the expression of other ECM proteins, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) involved in the pathogenesis of OA . Studies of cytokine effects on chondrocytes in vitro suggest that IL-4 alters the ratio of MMPs and TIMPs in favor of TIMPs by suppressing IL-1–stimulated MMP3 production . Integrin-regulated production of IL-4, as a result of optimal mechanical stimulation in normal articular cartilage in vivo, would be chondroprotective by inhibiting cartilage degradation and promoting matrix synthesis in normal articular cartilage. In contrast, in joint diseases such as OA, normal mechanotransduction pathways may be disrupted following changes in integrin expression by chondrocytes or neo-expression of adhesive and antiadhesive molecules such as fibronectin and tenascin in the pericellular matrix, resulting in abnormal chondrocyte activity. Indeed, preliminary data from our laboratory indicate that chondrocytes from OA cartilage show an abnormal electrophysiological response to both mechanical stimulation and direct application of IL-4 . Further elucidation of the signaling events activated by mechanical stimuli in HAC from normal and diseased cartilage should lead to a better understanding of how cartilage is maintained by mechanical stimuli in health and disease. These studies suggest that better understanding of the signaling molecules involved in mechanotransduction in chondrocytes may also lead to the identification of novel targets for therapy in OA. | Study | biomedical | en | 0.999996 |
10189378 | The wb alleles, wb k05612 , wb k00305 , wb PZ09437 , wb PZ10002 , wb SF25 , wb HG10 , and wb CR4 , were used to determine embryonic functions for the Wb protein. P element induced alleles, wb k05612 , wb k00305 , wb PZ09437 , and wb PZ10002 , were produced in the laboratories of Istvan Kiss and A. Spradling (Carnegie Institute, Baltimore, MD), and ethylmethane sulfonate (EMS) induced alleles, wb SF25 and wb HG10 , were produced in the laboratory of M. Ashburner (University of Cambridge, Cambridge, UK). wb SF25 , wb HG10 , and wb PZ09437 have been described previously . Df(2L)fn 7 (breakpoints 34E3; 35B3-4) and Df(2L)fn 36 (breakpoints 34F3-5; 35B4) were used in this study and are described in Lindsley and Zimm, . Revertants of wb PZ09437 were obtained by precise excision of the P element and showed wild-type appearance and fertility. Lethal chromosomes used in this study were kept in stocks balanced over CyO . Somatic clones in the eye were produced by inducing mitotic recombination using the FLP/FRT system as described in Roote and Zusman . Embryos from mutant lines were placed on petri perm plates (Hereus) in a drop of Voltalef 3S oil. All embryos were derived from mothers homozygous for the klarsicht ( kls ) mutation, which clears out yolk and makes embryonic phenotypes easily visible during filming, yet has no discernible effect on embryonic development . Time lapse videomicroscopy was performed on embryos under a Zeiss Axioskop microscope with a Panasonic AG-6730 recorder and a Zeiss ZVS-47N CCD videocamera system. wb embryos were identified by their inability to hatch and the presence of a dorsal hole at the end of embryonic development. Embryos were collected on agar/apple juice plates and prepared for immunostaining according to the protocol described in Zusman et al. with an antibody against a pericardial protein or an antibody against a tracheal protein . Embryos stained with antibodies were dehydrated and mounted in a 3:1 solution of methyl salicylate and Canada balsam for examination under bright-field illumination. For examination of somatic muscles, wb embryos were prepared as described by Drysdale et al. and viewed under polarized light. To confirm and examine further the wb somatic muscle phenotype, embryos derived from parents heterozygous for wb were stained with antibodies against muscle myosin using the procedures described in Young et al. and Roote and Zusman . Late stage wb or deficiency-containing embryos were identified by the dorsal hole phenotype and/or their inability to hatch. At earlier stages wb phenotypes were based on 25% of the population exhibiting defects not observed in a wild-type population, and the similarity of these defects to those observed when a dorsal hole is present. The mutant tracheal phenotype was also observed in developing wb embryos using videomicroscopy. Southern and Northern blot analyses were performed by standard procedures . RNA was extracted by the guanidium thiocyanate/phenol/chloroform extraction method of Chomczynski and Sacchi . Poly(A) + RNA was isolated using a Pharmacia Kit ( Pharmacia Biotech, Inc. ). Equal specific activity of wb probes and laminin α3, 5 and γ1 probes were achieved using a standardized labeling protocol, and by using probes of similar lengths and similar GC content. Exposure times for Northern blots were 3 d. Whole mount in situ hybridizations were conducted using digoxigenin labeled wb cDNAs following the protocol of Tautz and Pfeiffle . At least three sequence errors were discovered within the published DS 03792 sequence leading to reading frame shifts. Suitable cDNAs were isolated using PCR, subcloned, and were used to correct the derived cDNA sequence. Irregularities between the domain structure of vertebrate and this new Drosophila laminin α chain were confirmed by additional isolation of suitable cDNAs by PCR and subsequent sequencing, ruling out misleading interpretations of intron–exon boundaries. Two independent fragments from either the NH 2 or COOH terminus (amino acids 173–376 and amino acids 2,383–2,633, respectively) were cloned into the appropriate pMALc2 expression vectors (BioRad Laboratories). After induction and lysis of cells, fusion proteins were purified over a maltose matrix (BioRad Laboratories). Both antigens were used to generate two independent rabbit polyclonal antisera each. Polyclonal antisera were affinity purified over a corresponding GST fusion protein ( Pharmacia Biotech, Inc. ) column and eluted with 0.1 M glycine, pH 2.5. The specificity of the antisera was tested on Df(2L)fn 36 embryos. For histochemical staining, the antifusion protein antisera were used at a concentration of 1:500. Samples of embryonic extracts and conditioned medium of Schneider S2 cells were separated under nonreducing and reducing conditions on 6% SDS-PAGE. After transfer onto nylon membranes, blots were probed with anti- wb antibodies and detected with HRP conjugated secondary antibodies, followed by ECL chemiluminescence (Nycomed Amersham, Inc. ). In our attempt to find laminin-like sequences from Drosophila in the database, we noticed the presence of EGF-like repeats similar to laminin chains on the reverse strand of a subclone derived from the genomic phage DS 03792 . Subsequent alignment of all subclones derived from this DS phage revealed the presence of a novel laminin chain gene in Drosophila . Analysis of the gene structure showed a genomic region spanning ∼70 kb of DNA with ≥16 exons contained within two overlapping DS phages, DS 037092 and DS 01068 . Most intron–exon boundaries proposed by GENSCAN were confirmed by isolating and sequencing suitable cDNA clones spanning the region of interest (data not shown). Conceptual translation of the 10,101-nucleotide open reading frame yields a protein of 3,367 amino acids with a deduced molecular size of ∼374 kD . At the NH 2 terminus, the predicted initiating methionine is followed by an amino acid sequence containing structural regions characteristic of a secretory signal sequence . A hydropathy profile of the primary structure revealed no other long hydrophobic regions indicative of a transmembrane spanning segment , suggesting that this laminin chain is a secreted protein. Closer inspection of the domain structure shows that this new chain has all the domains of laminin α chains in the appropriate order . However, the number of different modules varies in some regions. For example, the second EGF-like stretch contains 10 full and 2 half EGF repeats, while in vertebrates there are 8 full and 2 half EGF repeats . In addition, a unique NH 2 -terminal extension of ∼120 amino acids is present . Finally, the array of the second EGF repeat region is symmetrically interrupted by an insertion of 45 amino acids. We performed domain-wise comparisons of identities to existing vertebrate α chains. The LN domain showed almost an equally high degree of identity to vertebrate α1 and α2 chains, while the LE4 domain showed a slightly higher degree of identity to vertebrate α2 than to α1. However, both L4 domains showed slightly higher scores of identity to α5, immediately followed by equally high scores to α2 and α1. The remaining two EGF-like repeats showed that the first was highly homologous to α1 but the second was homologous to α2. Finally, all five G domains showed a slightly higher similarity to α2 than to α1. In summary, the majority of the domains showed most similarity to vertebrate α2 chains, yet many were significantly similar to α1. For this reason, and to illustrate the fact this chain is a common precursor of vertebrate α2 and α1 chains, we have tentatively called this chain Drosophila laminin α1, 2 in the remainder of the text. A special feature within the amino acid sequence should be noted: the presence of a RGD within the first L4 domain . RGD tripeptides have been shown to mediate cell adhesion in Drosophila using Drosophila PS2 integrins as receptors . In fact, a recent study based on cell culture assays demonstrated that the laminin α1, 2 subunit showed exclusive binding to one integrin isoform, αPS2m8βPS4A, while the other PS2 integrin isoforms did not show any binding , suggesting that α1, 2 is a ligand of a splice-specific form of the PS2 integrins. Northern analysis was performed on RNA derived from samples spanning the Drosophila life cycle using α1, 2 cDNAs as probes. An 11-kb transcript was first detected in the early stages of embryogenesis and peaked in 6–12-h embryos . In the last part of embryogenesis (12– 18 h), a slightly smaller version of a 10.5-kb transcript was observed. We cannot exclude the possibility of an alternative spliced transcript or alternative usage of another polyadenylation site. Transcription decays in the later stages and is hardly detectable in third-instar larvae, but increases again in pupal stages. To compare the existing Drosophila laminin chains, the same Northern blot used for α1, 2 was also probed with a mixture of α3, 5 and γ1 probes . This showed that the two laminin subunits are present at similar stages during embryogenesis. There is a marked difference, however, as these two subunits are also transcribed very strongly during the late stages of embryogenesis, in contrast to α1, 2 which fades out rapidly during this stage. Assuming that all probes in this analysis had similar specific activities (see Materials and Methods), it suggests that α1, 2 is less abundantly expressed than α3, 5, a feature already noted in vertebrate expression studies . Using digoxigenin-labeled probes, the spatial expression of the α1, 2 chain was examined. Transcripts were first detected during oogenesis in nurse cells and growing oocytes , suggesting a maternal contribution. During cleavage stage, the message is uniformly distributed in the egg and becomes slightly enriched in cells of the trunk region at blastoderm stage . During germband extension , low levels of uniform expression are observed. After germband retraction, the visceral mesoderm of the gut starts to accumulate α1, 2 transcripts . At that time, cells near the presumptive muscle attachment sites show transcripts . At stage 14, strong expression is also observed in cardiac cells and more prominent in cells near the muscle attachment sites . Transcription of laminin α1, 2 is also readily detectable in imaginal discs, as assayed by LacZ staining of imaginal discs derived from the viable P element line H155 which mimics the embryonic transcript pattern faithfully (data not shown). Particularly strong expression was found in wing discs, where certain groups of cells in the presumptive wing dorsal and ventral region show LacZ staining . Strong staining was also observed in the eye antennal disc immediately behind the morphogenetic furrow , and also in a specific pattern in leg discs . To assess the nature and appearance of the α1, 2 protein, polyclonal antisera against the NH 2 and COOH termini (see Materials and Methods) were produced and assayed both by Western analyses and on whole mount embryos. Western blotting of conditioned medium of Schneider S2 cells showed a single 360-kD band , while in embryonic extracts proteolytic cleavage was observed giving rise to a 240-kD band (lanes 4 and 5) and a 110-kD band (lane 5) which are detectable using anti-NH 2 and -COOH antibodies, respectively. This suggests that proteolytic cleavage also occurs in Drosophila , as was reported for the vertebrate α2 chain . NH 2 antibodies also detected a possible further degradation product of ∼180 kD (lane 4), which is not detected by COOH antibodies. Both antisera recognize a single 800-kD band under nonreducing conditions (lanes 1 and 2), suggesting that the α1, 2 protein is part of a laminin trimer. Using an immunoprecipitation assay, α1, 2 was found to be associated with the same β and γ chains, as was α3, 5 (data not shown). The protein is first detected at stage 10 as a weak diffusive stripe between the ectoderm and the mesoderm . During germband retraction the protein is localized diffusely around areas that constitute the visceral mesoderm. At stage 14, strong staining is observed in the BMs that surround the digestive system, i.e., the gut , or at muscle attachment sites . These patterns are strongly reminiscent of the expression patterns of various Drosophila integrin subunits, particularly the β subunit and the α2 subunit . Later stages include localization in dorsal structures along the ventral nerve cord , and BMs around the digestive system . During imaginal wing disc development, α1, 2 is localized in a specific spot pattern on the presumptive wing dorsal and ventral region . Genomic phage DS 03792 was mapped to chromosomal region 35A1 . Several P element insertion events could be detected within the genomic area of the laminin gene. Of particular interest were two fly lines conferring embryonic lethality that showed the P element inserted into the middle of the fourth intron . Because insertions of this type showed lethality on other occasions where an unusual splicing event was shown to be the cause for lethality, we wondered whether the same situation would apply here. To test whether trans-splicing between the fourth exon of laminin α1, 2 and the last exon of ribosomal protein S12, which resides on the P element construct, we performed Northern analysis on RNA derived from l(2) 09437 embryos or l(2) 10002 embryos (not shown). Two bands were visible: the doublet band ∼11 kb, already detected in the developmental Northern analysis generated by the wild-type gene from the balancer chromosome, and a smaller species of 5.6 kb, derived from the mutant chromosome whose RNA showed trans-splicing to S12, yielding a shorter transcript . Rehybridization of the same Northern lane using a S12-specific probe confirmed the same 5.6-kb mRNA species (data not shown). We interpret the fact that the 5.6-kb mutant band is stronger than the wild-type 11-kb band as a composite result of a higher efficiency to complete the transcript, because the mutant transcript is more stable, or the transfer of larger mRNA is less efficient. The shortened mRNA codes for a protein truncated within LE8 , and as a result no assembly of the heterotrimeric laminin molecule can occur, as only α, β, and γ heterotrimers are sufficiently stable to be secreted . Consequently, it is likely that no functional laminin of the subunit composition α1, 2; β1; γ1 is secreted in the l(2) 09437 mutant. A survey in the chromosomal area of 35A showed that a locus, termed wb , resulting in blisters in wings, could account for the loss of laminin function. To test this hypothesis, l(2) 09437 and l(2) 10002 flies were crossed to suitable viable and embryonic lethal wb alleles, and were tested for complementation. None of the strong ethylmethane sulfonate induced wb alleles complemented l(2) 09437 and l(2) 10002 for embryonic lethality (data not shown). This result, in combination with the mapping data, strongly argues that l(2) 09437 and l(2) 10002 are mutations in the wb locus, and that wb is indeed laminin α1, 2. To examine the functions of the Wb protein during embryogenesis, the development of embryos homozygous for embryonic lethal mutations in the wb gene was examined and compared with wild-type embryos and embryos homozygous for a deficiency that uncovers the wb locus . Time lapse videomicroscopy of developing flies revealed that homozygous wb HG10 , wb k05612 , and Df(2L)fn 36 embryos become abnormal during gastrulation. Rather than extending their germbands dorsally, mutant germbands twist and extend laterally . Near the completion of germband extension, wb and Df(2L)fn 36 embryos show a distinct separation between the mesodermal and ectodermal tissue layers of the germband . These phenotypes are similar to that described for mys hemizygous embryos which lack the β PS subunit of integrin , a potential receptor for laminin . Another phenotype in common with mys embryos includes a dorsal hole which often forms in the cuticle of wb k05612 , and occasionally in wb k00305 embryos. Although Wb protein accumulates around the BMs of the developing embryonic gut, no defects were detected in gut morphology or midgut primordial migration. Previous studies of embryos lacking the Drosophila laminin α3, 5 chain have demonstrated functions for this molecule in the proper morphogenesis of heart, somatic muscle, and trachea . In laminin α3, 5 deficient embryos there is a dissociation of the pericardial cells of the heart, gaps in the dorsal trunk of the trachea, and the ventral oblique muscles fail to reach their attachment sites. Similar heart and tracheal defects are found in embryos with mutations affecting α PS3 β PS integrin . To determine if the Wb protein is also involved in these processes, we examined the development of their heart, trachea, and somatic muscles. The heart (dorsal vessel) forms from external pericardial cells and internal cardioblasts that migrate during dorsal closure to meet along the dorsal midline to form the heart tube . wb and wb -deficient embryos stained with antibodies that recognize pericardial cells show that homozygous wb k05612 , wb k00305 (both occasionally showing dorsal holes), and Df(2L)fn 36 embryos often contain fewer pericardial cells than wild-type embryos resulting in distinct gaps in the heart tube . Furthermore, the pericardial cells appear to dissociate randomly and the tube often appears to curve off towards the lateral side of the embryo. The dorsal trunk of the trachea is formed by migration of the tracheal pits to form a long tracheal tube which extends the length of the embryo . Antibodies were used to examine trachea formation in wb and wb deficient embryos. Embryos homozygous for wb k05612 , wb HG10 , and Df(2L)fn 36 were observed to have significant gaps in the dorsal trunk of the trachea . This was confirmed by examining the development of filmed wb embryos. Due to the strong expression of the Wb protein in muscle attachment sites, we also examined wb and wb -deficient embryos for defects associated with the attachment of myotubes to their ectodermal attachment sites. Careful examination of somatic muscle in homozygous wb k05612 , wb HG10 , and Df(2L)fn 36 embryos stained with antimyosin antibodies , or prepared for examination under polarized light at the end of embryonic development, revealed that their somatic myotubes are often not attached to target epidermal attachment sites. This defect most commonly involves the ventral oblique muscles located in the anterior most segments of the embryo . Random disorganization of myotubes and areas without myotubes are occasionally observed in these embryos as well. In conclusion, several defects are observed in wb embryos, some in common with those observed in laminin α3, 5 embryos, and many in common with those observed with integrin mutations. As the name implies, mutations in the wb locus can lead to blistering of the wing, in which the dorsal and ventral wing surfaces separate . As shown in Fig. 8 , A and C, the blisters are located centrally within the wing, consistent with the location of laminin expression and localization in wing discs . The blisters vary in size, depending on the allelic combination used (data not shown). Homozygous viable alleles of wb exist that show no blistering (i.e., wb CR4 ), and only in combination with an embryonic lethal wb allele (i.e., l(2) 09437 ) or a deficiency ( Df[2L]fn 7 or fn 36 ) were blisters observed, suggesting that below a certain threshold, the lack of functional laminin can lead to blistering. No haplo-insufficiency was observed in l(2) 09437 animals (data not shown). The wb phenotype strongly resembles the phenotypes associated with mutations in integrins , and mutations in the Drosophila laminin α3, 5 gene can also lead to blistered wings . Due to the fact that high expression of wb was also found posterior to the morphogenetic furrow in the developing eye , we wished to determine the function of wb during eye development. For this reason, somatic clones were induced in the eye of wb k05612 flies using the FLP technique . As evident in Fig. 8 D, the number of photoreceptor cells did not change, but they appear disorganized. Disorganized photoreceptor cells were also detected in mys and mew (PS1-encoding) mutant clones . We have demonstrated the existence of a second laminin α chain in Drosophila , and sequence analysis shows that it is homologous to the α2 and α1 chain in vertebrates. Most likely, this chain represents one of the ancestral versions of a vertebrate α chain of laminin, as some marked changes are observed in comparison to α1, 2. The protein is slightly larger than vertebrate α1 or α2, mainly due to the addition of a NH 2 -terminal extension, an insertion in the first EGF-like region, and by acquisition of two additional EGF-like modules . Other discrepancies have been observed in the Caenorhabditis elegans α1, 2 where one G module is deleted . Laminins have also been isolated in lower organisms such as Hydra vulgaris where they are expressed in the subepithelial zone involved in attachment of mesoderm to the ectoderm. Sequence comparisons suggest that the α chain associated with this laminin corresponds to an ancestral version of the α3 and α5 chain (Sarras, M., personal communication). Virtually no exon boundaries match the gene structure observed in human laminin α2 or C . elegans laminin α1, 2, nor is the number of exons similar , suggesting that α chains in higher animals have become more complex by splitting coding sequences through uptake of new noncoding sequences. In addition, no exon boundaries of Drosophila α1, 2 fit those of Drosophila α3, 5 or even of C . elegans α1, 2 , suggesting that the two α chains diverged much earlier. Based on the sequenced C . elegans genome, which discovered only two α chains, it is plausible to assume that invertebrate genomes such as Drosophila or C . elegans probably possess only two α chains, one β and one γ chain, respectively, which may limit the number of possible assemblies into functional laminin trimers to two. A comparison between expression patterns of α1, 2 and vertebrate laminins reveals that the expression of vertebrate α2 fits better to Drosophila α1, 2, as α1 shows a highly restricted expression in kidney, as compared with α2 whose expression was reported to be widespread in mesenchymal cells . In accordance with vertebrate expression studies where α5 was shown to be the most widely expressed α chain, Drosophila α3, 5 is more widely expressed than α1, 2. Interestingly, Wb harbors a RGD sequence located on the L4 domain which makes it a likely ligand for integrins. Biochemical studies on integrin-mediated adhesion using Drosophila cell lines identified Wb as a distinct ligand for αPS2m8βPS4A integrin , one of four splice forms of the αPS2βPS integrins . The αPS2 isoform is also the predominant splice form present at developmental stages during which Wb is expressed . No data have been reported to date on the isoform distribution of βPS integrin. In contrast, other RGD containing proteins such as tiggrin , or ten-m show no absolute requirement for a specific splice isoform of βPS: both proteins need only exon 8 of αPS2 to be present. Using a similar approach, Drosophila laminin containing α3, 5 was shown to be a specific ligand for αPS1βPS integrin . This suggests that Drosophila laminins (subunit composition α1, 2; β1; γ1, and α3, 5; β1; γ1) can serve as PS2 and PS1 integrin ligands, respectively. Moreover, the model for embryonic muscle and pupal wing attachment proposed by Gotwals et al. holds true, by juxtaposing another partner to tiggrin facing the PS2 binding site. Interestingly, the region harboring the RGD in L4 of Wb is highly related to the RGD-containing site of vertebrate laminin α5 , which could indicate that vertebrate α5 has taken up this motif during evolution, in contrast to the existing Drosophila α3, 5 which does not harbor an RGD site. Genetic data further support an association of wb with integrins, since weak mys mutations increase the size and frequency of blisters in wb flies (Khare, N., and S. Baumgartner, manuscript in preparation). No conclusive genetic interaction data were reported to occur between α3, 5 and mys . Several embryonic wb phenotypes were shown to be remarkably similar to those of single integrin mutations, i.e., the separation of mesoderm and ectoderm, and the twisted germband common to mys or to scb . Notably, separated mesoderm/ectoderm and twisted germband were not observed in mutations in the α3, 5 chain . The α3, 5 chain was only found to be required for later stages of patterning of mesodermally derived cells, suggesting that α1, 2 is exclusively used to confer early adhesion between mesoderm and ectoderm. In contrast, common phenotypes between α1, 2 and α3, 5 were detected in late stages of embryogenesis where the formation of the ventral oblique muscles is disturbed, particularly in the anterior segments . Finally, the formation of the heart was reported to be disturbed in mutations of both genes . No phenotype reminiscent of the muscular dystrophy-like phenotype in vertebrates was observed in our mutants. Although we did not observe wb expression in muscles, we cannot rule out marginal expression levels below the sensitivity of our detection method. However, certain myotubes do appear disorganized in wb mutant embryos. This cannot be considered an analogous situation to dy/dy mice , because the defects observed are most likely due to the inability of muscle cells to migrate properly and a failure in attaching to muscle attachment sites. Similar phenotypes were also observed in laminin α3, 5 mutants . Previous studies have shown that integrin-mediated adhesivity between the two epithelial cell layers of the wing is particularly sensitive to mutations involving either integrin ligands (this paper) or upstream factors of integrins, i.e., the blistered ( bs ) gene encoding a Drosophila serum response factor . bs and integrins interact genetically and mys expression appears to be greatly reduced in hypomorphic bs mutants , suggesting a scenario where bs might directly control integrin gene expression on the transcriptional level. It is plausible to assume that bs might also directly control wb expression, as the transcript pattern of both show striking coexpression , and a corresponding SRE has been located 260 bp upstream of the putative TATA box of the wb gene (data not shown). Specific screens have been performed for mutations affecting adhesion between wing surfaces . To our surprise, none of the loci described correspond to wb , suggesting that the formation of blisters in the wing depends on subtle changes of wb activity. This is further suggested by the fact that only suitable wb allelic combinations show blisters. For example, blisters were only detected in transheterozygous allelic combinations of a weak (homozygous viable) allele, wb CR4 , and Df(2L)fn 7 or l(2) 09437 which behaves as a null allele. In other words, only the range of wb activity slightly below 50% of wild-type activity is capable of forming blisters, while a level of ≥50% does not affect wing blistering, as no haplo-insufficiency is observed in l(2) 09437 flies. In parallel to the wing, wb clones induced in the eye cause similar phenotypes to clones induced in integrin mutations, i.e., αPS1 ( mew ) mutants or βPS ( mys ) mutants , but not in αPS2 ( if ) mutants which result in virtually wild-type eyes. Similar phenotypes were also observed in laminin α3, 5 mutant combinations, however, the degree of severity of disorganization is higher than in wb or integrin mutant clones . | Study | biomedical | en | 0.999995 |
10190893 | CD40-deficient mice have been described and were originally provided by Dr. H. Kikutani (Osaka University, Osaka, Japan ). As control mice, CD40 +/− or C57BL/6 mice were used, giving similar results. LCMV (WE strain) was grown on L cells at a low multiplicity of infection. LCMV WE was originally provided by Dr. R. Zinkernagel (University of Zürich, Zürich, Switzerland). Influenza virus (strain PR8) was originally provided by Dr. J. Pavlovic (University of Zürich) and grown in day 10–fertilized chicken eggs. Mature bone marrow–derived DCs were generated as described ( 24 ). Anti–IL-12 p35 (C18.2) and p40 (C15.1) mAbs were provided by G. Trinchieri (Wistar Institute, Philadelphia, PA ). For activation of T cells in vitro, T cells were purified and stimulated with anti-CD3 and anti-CD28 Abs as described previously ( 24 ). Soluble TRANCE produced from recombinant baculoviruses has been described previously ( 24 ). TRANCE-R–Fc (TR-Fc), a recombinant protein of the extracellular domain of TRANCE-R fused to the constant region of human IgG1, was produced in a similar way using a baculovirus system and purified on protein A–Sepharose beads ( Amersham Pharmacia Biotech ). For LCMV-specific CD4 + T cell proliferation, mice were infected intravenously or into one hind footpad with 200 PFU of LCMV WE. Spleen cells were isolated 13 or 30 d later, and proliferation and cytokine production were measured as described ( 19 ). To assess cytotoxicity, mice were infected intravenously with 200 PFU of LCMV, and spleen cells were isolated 8 d later. For influenza virus–specific proliferation, mice were infected intranasally with virus (0.1 hemagglutination U/mouse). Spleen cells were isolated 8 d later. Mice were injected three times, on days 0, 2, and 5 after infection, with 100 μg of either TR-Fc or control hIgG1. EL-4 target cells were pulsed with peptide p33 (KAVYNFATM) at a concentration of 10 −7 M for 90 min at 37°C in the presence of [ 51 Cr]sodium chromate in IMDM supplemented with 10% FCS. Cells were washed three times, and 10 4 cells were transferred to a well of a round-bottomed 96-well plate. Stimulated or ex vivo–isolated spleen cell suspensions were serially diluted and mixed with peptide-pulsed target cells. Plates were centrifuged and incubated for various time spans at 37°C. At the end of the assays, 70 μl of supernatant was counted in a γ-counter. Spontaneous release was determined by adding medium instead of effector cells, and total release was determined by adding 2 M HCl instead of effector cells. Percent specific release was calculated as 100 × (experimental release − spontaneous release)/(total release − spontaneous release). For assessment of B cell responses, LCMV-specific IgG Abs were determined as described on plates coated with LCMV nucleoprotein produced by recombinant baculoviruses ( 19 ). PNA staining was performed on acetone-fixed frozen sections as described ( 28 ). For LCMV-specific CD4 + T cell proliferation, spleen cells were isolated 13 or 30 d after infection and CD4 + T cells were purified by MACS ® according to the instructions of the supplier (Miltenyi Biotech). Purity was >95%. 10 5 CD4 + T cells were stimulated with 10 5 irradiated LCMV (highest concentration = multiplicity of infection = 0.3) or peptide 13 (GLNGTDIYKGVYQFKSVEFD; highest concentration = 3 μg/ml)–pulsed splenic APCs, and proliferation was assessed 3 d later by [ 3 H]thymidine incorporation. Production of IFN-γ was assessed in the wells with the highest antigen concentration by ELISA ( 19 ). For influenza virus–specific CD4 + T cell responses, spleen cells were isolated 8 d after infection, and purified CD4 + T cells (2 × 10 5 cells/well) were restimulated with irradiated spleen cells (10 5 cells/well) in the presence of various concentrations of UV light–inactivated, purified influenza virus. Proliferation and IFN-γ production were measured as described above. A recently identified member of the TNF receptor family, TRANCE-R (also called RANK), has been shown to be expressed at high levels on mature DCs ( 23 – 25 ). Moreover, TRANCE treatment enhanced the survival of mature DCs, indicating that TRANCE-R may exhibit a similar function as CD40 on these cells ( 24 ). To test whether TRANCE/ TRANCE-R interaction may play a role in T cell activation, surface expression of TRANCE was analyzed on activated T cells. Similar to CD40L, surface TRANCE expression was highly upregulated on T cells upon stimulation through antigen receptors . Moreover, when mature DCs were treated with soluble TRANCE, the expression of IL-12 and other inflammatory cytokines (e.g., IL-1 or IL-6; data not shown) was induced in mature DCs, a property also shared by CD40L ( 11 – 13 ). Together, these results suggested that TRANCE and CD40L may share some similar functions in vivo during T cell activation and that TRANCE may be responsible for CD40L-independent CD4 + T cell responses, as observed in some murine model systems such as during viral infections ( 19 – 21 ). To test this hypothesis, we chose to study the immune response to LCMV infection as a murine model since it has been extensively characterized and also because the activation of CD4 + T cells during LCMV infection was shown not to be affected in CD40L- or CD40-deficient mice ( 19 ). To analyze whether TRANCE is upregulated in vivo during the course of an immune response after viral infection, mice were infected with LCMV, and spleen cells were analyzed for TRANCE expression 8 d later. Indeed, the proportion of TRANCE-expressing T cells increased after infection (∼6% of CD4 + T cells and ∼7% of CD8 + T cells became TRANCE-positive, whereas 0% of T cells expressed TRANCE in uninfected control mice). To determine whether TRANCE plays a role during immune responses in vivo, and if so, whether it exhibits a compensatory role for CD40L during viral infections, we tested the consequences of blocking the TRANCE/TRANCE-R interaction by injection of TR-Fc on antigen-specific B, CD8 + , and CD4 + T cell responses induced by LCMV infection in control (C57BL/6 or CD40 +/− ) and CD40-deficient mice ( 26 ). The most prominent role of CD40L is to promote isotype switching in activated B cells and to allow the formation of germinal centers (GCs; 1–3). Indeed, CD40-deficient mice failed to produce high titers of LCMV-specific IgG Abs and produced no GCs . In contrast, TR-Fc–treated C57BL/6 mice mounted LCMV-specific IgG responses comparable to those of control mice treated with hIgG1 and generated similar numbers of GCs of normal architecture . These results suggest that the TRANCE/TRANCE-R interaction does not play a critical role in T–B cell collaboration, despite the low level of TRANCE-R that can be detected on activated B cells (data not shown). We next analyzed the ability of TR-Fc–treated control and CD40-deficient mice to mount LCMV-specific T cell responses. Mice were injected with LCMV, and CD8 + T cell–mediated responses were analyzed in a 51 Cr-release assay 8 d later . In keeping with previous reports ( 20 , 21 ), the CD40L/CD40 interaction was not required for efficient primary CTL responses against LCMV . In addition, inhibition of the TRANCE/TRANCE-R interaction did not affect the LCMV-specific acute CTL responses . Moreover, inhibition of both the TRANCE/ TRANCE-R and CD40L/CD40 interactions did not affect acute CTL responses . These results suggest that primary LCMV-specific CTL responses are largely independent of CD40L and TRANCE on activated T cells. LCMV-specific CD4 + T cell responses were then examined early after infection (day 13) in TR-Fc–treated control and CD40-deficient mice by measuring in vitro recall proliferative responses. As reported previously ( 19 ), LCMV-specific CD4 + T cells produced a Th1 cytokine pattern, since large amounts of IFN-γ but not IL-4 (data not shown) were detected in culture supernatants. Purified CD4 + T cells from CD40-deficient mice proliferated normally and produced, although at reduced levels, IFN-γ after stimulation with LCMV-derived antigens , indicating that LCMV can prime antigen-specific CD4 + T cells in a CD40L/CD40-independent manner. TR-Fc–treated control mice also mounted normal CD4 + T cell responses . In marked contrast, the proliferative response of CD4 + T cells in TR-Fc–treated CD40-deficient mice was nearly completely blocked . In addition, the production of IFN-γ was also completely abrogated in these mice . This was not due to immune deviation, since blocking the TRANCE/TRANCE-R interaction in control or CD40-deficient mice did not upregulate IL-4 production (data not shown). To determine whether the lack of LCMV-specific CD4 + T cell responses was due to a delay in T cell priming in the absence of both CD40L/ CD40 and TRANCE/TRANCE-R interactions, CD4 + T cells were purified and restimulated with viral antigens 1 mo after infection. Even 30 d after infection, no significant LCMV-specific CD4 + T cell responses were detected in the absence of both CD40L/CD40 and TRANCE/ TRANCE-R interactions . Therefore, the results indicate that either the CD40L/CD40 or the TRANCE/ TRANCE-R interaction is required for induction of CD4 + T cell responses by LCMV. To analyze whether TRANCE can also mediate CD40L/ CD40-independent CD4 + T cell responses in other viral systems, CD40-deficient mice were infected with influenza virus, and virus-specific CD4 + T cell responses were analyzed . As observed for LCMV, influenza virus can prime antigen-specific CD4 + T cells in a CD40L/CD40-independent manner, and the induction of virus-specific CD4 + T cell responses was greatly inhibited in the TR-Fc–treated CD40-deficient mice . Thus, TRANCE/TRANCE-R provides a major costimulatory stimulus in the absence of CD40L/CD40 for CD4 + T cell responses to influenza viruses. In summary, this study establishes the TRANCE/ TRANCE-R interaction as an important player in CD4 + T cell responses in vivo. Moreover, we also show that the TRANCE/TRANCE-R interaction compensates for a lack of CD40L/CD40 interaction to allow efficient CD4 + T cell responses during viral infection. This explains why viruses can induce CD4 + T cell immune responses in CD40L- or CD40-deficient mice. In addition, this study also shows that despite the destruction of infected cells and the production of various inflammatory cytokines in response to viral infection, efficient CD4 + T cell priming still requires costimulation predominantly by TNF family members (i.e., either TRANCE or CD40L), which is analogous to CD4 + T cell priming induced by purified proteins administered with CFA (in this case, costimulation provided by CD40L). Therefore, it is possible that CD4 + T cell priming in general may require costimulation by at least one TNF family member (e.g., CD40L or TRANCE). CD40L-mediated CD4 + T cell activation occurs indirectly via activation of the APCs ( 1 – 3 , 9 – 13 ). Specifically, in vitro stimulation of CD40 on DCs stimulates a maturation process culminating in the upregulation of costimulatory molecules and the capacity to produce IL-12, a cytokine important for production of IFN-γ by CD4 + T cells ( 1 – 3 , 9 – 13 ). Although stimulation of TRANCE-R on mature DCs fails to upregulate costimulatory molecules on these cells ( 24 ), we showed that, similar to CD40L, TRANCE treatment triggered generation of IL-12 and other proinflammatory cytokines by mature DCs. In addition, when stimulated in vitro by anti-CD3, purified T cells proliferated and produced normal levels of cytokines in the presence of TR-Fc (data not shown), suggesting that, similar to the CD40L/CD40 interaction, there is no direct role for the TRANCE/TRANCE-R interaction in T cells. Therefore, the TRANCE/TRANCE-R and CD40L/CD40 interactions between CD4 + T cells and APCs may have functional consequences primarily for the APCs, e.g., promoting DC viability and cytokine production ( 1 – 3 , 9 – 13 , 24 ). It is presently not known why some antigens (e.g., proteins in adjuvants) use predominantly the CD40L-dependent pathway ( 1 – 3 ) while others (e.g., viruses [this study]) use both TRANCE- and CD40L-dependent pathways of CD4 + T cell stimulation. It is possible that certain viruses directly upregulate TRANCE-R during DC differentiation. Alternatively, there may be different requirements for induction of TRANCE and CD40L on T cells. It is also possible, although not yet determined, that some pathogens might use predominantly the TRANCE-dependent pathway to elicit efficient CD4 + T cell responses. The CD40L/CD40 interaction is an important site for manipulating the immune response in order to facilitate organ transplantation and to reduce atherosclerosis ( 1 – 3 , 29 ), and our in vivo findings now suggest that the interaction of TRANCE and its receptor may be an additional target for immunotherapy. | Study | biomedical | en | 0.999996 |
10190894 | Female SWXJ (H-2 q,s ) mice were bred at the Biological Resources Facility of the Lerner Research Institute by mating SWR/J (H-2 q ) females with SJL/J (H-2 s ) males purchased from The Jackson Laboratory . Mice were immunized at 7–12 wk of age. All protocols for animal research met with prior approval of the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic Foundation in compliance with the Public Health Service policy on humane care and use of laboratory animals. The PLP peptides 104–117 KTTICGKGLSATVT, 139–151 HSLGKWLGHPDKF (serine for cysteine at residue 140), and 178–191 NTWTTCQSIAFPSK, as well as MBP 87–99 VHFFKNIVTPRTP and MOG 92–106 DEGGYTCFFRDHSYQ, were either purchased commercially (Bio-Synthesis) or synthesized at the Protein Core Facility of the Lerner Research Institute with standard solid phase methodology using amino acids with 9-fluorenylmethoxycarbonyl (FMOC) side chain protection. Peptides were purified >90% by reverse phase HPLC using a 22 × 250 mm C-18 column (Vydac Separations Group). The identity of each purified peptide was confirmed by mass spectrometry. A PLP pin peptide series representing a walk-through of the entire 276-amino acid primary sequence of mouse PLP ( 11 , 12 ) was purchased from Chiron Mimotopes. A total of 265 overlapping 12-mers were synthesized on high-density polyethylene rod tips assembled into holders designed in 96-well microtiter plate format ( 13 ). Each successive peptide differed from the previous 12-mer by sequential NH 2 -terminal deletion and COOH-terminal addition of PLP amino acids. Upon arrival, 1 mg of each PLP pin peptide was dissolved in 500 μl of a solution of 40% acetonitrile ( Aldrich Chemical ) in 10 mM Hepes buffer ( GIBCO BRL ). Working aqueous concentrations of pin peptides were prepared at 150 μg/ml in PBS, pH 7.2, and 20 μl of each working solution was distributed sequentially into individual wells of 96-well flat-bottomed microtiter Falcon plates ( Becton Dickinson ). The plates were stored at −20°C until ready for use. EAE was induced as previously described ( 14 ). SWXJ mice were immunized by subcutaneous injection in the abdominal flanks on day 0 with 100 nmol of PLP peptides p139–151 (154 μg), p178–191 (158 μg), or p104–117 (138 μg), and 400 μg Mycobacteria tuberculosis H37RA (Difco Labs.) in 200 μl of an emulsion of equal volumes of water and IFA (Difco Labs.). On days 0 and 3 each mouse also received intravenously 6 × 10 9 Bordetella pertussis bacilli (Michigan Department of Public Health, Lansing, MI). In the study presented here, all mice developed clinical EAE within 24 d of immunization. All mice were weighed and examined daily for neurologic signs as previously described ( 14 ) according to the following criteria: 0, no disease; l, decreased tail tone or slightly clumsy gait; 2, tail atony and/or moderately clumsy gait and/or poor righting ability; 3, limb weakness; 4, limb paralysis; 5, moribund state. The presence of relapse was determined when mice showed an increase in observed neurologic disability of at least one clinical score unit. The increased neurologic deficit was typically accompanied by an abrupt and substantial (>7%) weight loss. Brains and spinal cords were fixed in 10% phosphate-buffered formalin, and paraffin-embedded tissue sections were cut (10 mm each) for immunostaining as previously described ( 15 , 16 ). Sections were pretreated with 0.04% OsO 4 and 1% H 2 O 2 in 10% Triton (Electron Microscopy Sciences) and blocked with 5% normal goat serum (Vector Labs.) and 5% nonfat dehydrated milk for 60 min. Sections were treated sequentially with PLP monoclonal IgG 2a antibody (Harlan) at a 1:200 dilution for 14 h at 4°C, biotinylated goat anti–mouse IgG 2a (Southern Biotechnology Associates) at a 1:500 dilution for 30 min at 22°C, and avidin-peroxidase complex (Vector Labs.) for 1 h at 1:1,000 dilution. Sections were then treated with diaminobenzidine and 0.01% H 2 O 2 for 8 min, 0.04% OsO 4 for 30 s, and washed in PBS. Images were digitized using the AlphaImager 2000 System (Alpha Innotech) at 640 × 480 pixel resolution. Images were captured at 10× magnification with the black level scale set at 0, white level scale at 255, and gamma level scale at 1.0. All images were normalized by adjusting background gray matter stain to the same mean intensity value using Adobe Photoshop (Adobe Systems). The presence of demyelination in CNS meninges and parenchyma was determined visually as well as by digitized image analysis using NIH image software (version 1.57; National Institutes of Health, Bethesda, MD). At wk 2, 4, 8, 12, and in some cases 16 after immunization of SWXJ mice with a PLP determinant, splenocytes were tested for proliferative responses to PLP determinants p104–117 ( 14 , 17 ), p139–151 ( 18 ), and p178–191 ( 19 ), as well as MBP 87–99, an immunodominant encephalitogenic determinant for both SJL/J ( 20 , 21 ) and SWR/J ( 22 ) mice, and MOG 92–106, an immunodominant encephalitogen for SJL/J mice ( 23 ). Mononuclear cells were purified by centrifugation on Lympholyte-M (Accurate Chemical Co.) for 20 min at 2,500 rpm. Cells collected from the interface were washed three times in HBSS and resuspended in DME ( GIBCO BRL ) supplemented with 10% FBS (Hyclone Labs.), 2 mM fresh l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 30 mM Hepes buffer ( GIBCO BRL ), and 5 × 10 −5 M 2-ME. Triplicate test cultures containing peptide at 20 μg/ml were evaluated at 3 × 10 5 splenocytes/well in a total volume of 200 μl in 96-well flat-bottomed microtiter Falcon plates ( Becton Dickinson ). Triplicate positive control wells contained mouse CD3 mAb at 5 μg/ml ( PharMingen ), or 0.005 tuberculin units/ml of tuberculin purified protein derivative (PPD; Connaught), or 20 μg/ml Mycobacteria tuberculosis H37RA (Difco). Dose responses to whole PLP (0.1–100 μg/ml) were also assessed in each experiment. The PLP was prepared from a washed total lipid extract of bovine white matter ( 24 ) and was purified and converted to aqueous as previously described ( 14 ). Negative control wells contained no peptide. Cultures were incubated at 37°C in humidified air containing 5% CO 2 . At 4 d, cultures were pulsed with methyl-[ 3 H]thymidine (l.0 μCi/well, specific activity 6.7 Ci/mmol; NEN), and the cells were harvested after 16 h by aspiration onto glass fiber filters. Levels of incorporated radioactivity were determined by scintillation spectrometry. Results are expressed as stimulation index (SI) defined as mean cpm of triplicate experimental cultures with Ag divided by mean cpm of cultures without Ag. Mononuclear cells were recovered from the CNS at various clinical stages of EAE according to the method of Ford et al. ( 25 ). In brief, SWXJ EAE mice were killed by CO 2 inhalation and perfused with 20 ml of HBSS to remove hematogenous leukocytes. Brain tissue was teased and digested with 1 mg/ml of collagenase D ( Boehringer Mannheim ) and 50 Kunitz U/ml of DNase ( Sigma Chemical Co. ) at 37°C for 60 min. After washing, cells were resuspended in Percoll ( Amersham Pharmacia Biotech ) adjusted with HBSS to a specific gravity of 1.030 and layered on Percoll/ HBSS at a specific gravity of 1.095. After centrifugation for 30 min at 1,250 g , cells were removed from the interface, washed, and counted for total yield. Percentages of T cells (FITC-labeled anti-CD3 or anti-CD4; PharMingen ) and microglia (FITC-labeled anti-CD11b; PharMingen ) were determined by flow cytometry analysis. Proliferation assays in response to peptides were performed as described above using 2 × 10 5 CNS harvested cells/microtiter well. We have previously reported that monocentric monophasic IMDS patients with no evidence by history or exam of prior subclinical CNS disease typically show fully sustained proliferative responses over a 1-yr period to defined PLP regions ( 26 ). The development and progression of myelin self-recognition was further evaluated over a total period of 3 yr in three of the original monocentric monophasic IMDS patients: VS, a 28-yr-old female with inflammatory internal capsule syndrome; DL, a 21-yr-old female with partial transverse myelitis; and JB, a 34-yr-old male with acute brainstem syndrome. During the course of the study reported here, both VS and DL showed progression to CDMS at 154 and 60 wk, respectively, after initial onset of their neurologic symptoms. Thus far, JB has not shown progression to CDMS. The three monocentric monophasic IMDS subjects were seen by the study neurologist (R.P. Kawczak) within 2 wk of the onset of their neurologic symptoms and were repeatedly examined over the course of the current study for the evaluation of new symptoms. Disease activity in IMDS patients was defined as the occurrence of interval clinical or MRI activity as described previously ( 26 ). CDMS was defined as a relapse involving an anatomically different area of the nervous system compared with onset symptoms ( 27 ). Magnetic resonance imaging (MRI) was performed as described previously ( 26 ) in a 1.5 Tesla superconducting whole body imaging system (Siemens Medical System). Image analysis was performed by two neuroradiologists blinded to the clinical disposition of the patients. All patients were able to understand informed consent and comply with the study protocol approved by the Institutional Review Board of the Cleveland Clinic Foundation. PBMCs from each IMDS subject were serially evaluated for proliferative responses to the 265 overlapping PLP epitope-mapping peptides as previously described ( 26 ). PBMCs were separated by centrifugation on Ficoll-Paque ( Amersham Pharmacia Biotech ), washed three times in HBSS ( GIBCO BRL ), and resuspended in serum-free HL-1 media (Hycor) supplemented with 2 mM fresh l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 30 mM Hepes buffer ( GIBCO BRL ). Each well contained 10 5 PBMCs with 15 μg/ml pin peptide in a total volume of 200 μl. Triplicate positive control wells contained human CD3 mAb at 10 μg/ml (Ortho Biotech), tetanus toxoid at 1:1,000 dilution (Lederle Laboratories), 0.005 tuberculin U/ml of tuberculin purified protein derivative (PPD; Connaught), or 20 μg/ml Mycobacteria tuberculosis H37RA (Difco Labs.) although negative control wells contained either no peptide or one of 30 irrelevant pin peptides of myohemerythrin, a protein having minimal sequence homology with myelin proteins ( 28 ). Dose responses to whole bovine PLP (0.1–100 μg/ml) were also assessed in each experiment. Cultures were incubated at 37°C in humidified air containing 5% CO 2 . At 72 h, cultures were pulsed with methyl-[ 3 H]thymidine (NEN) and harvested and processed as described above. Test wells containing 15 μg/ml of a single PLP peptide were considered positive with an SI > 2.0 and with a Δcpm > 1,000 and at least three standard deviations above the mean of nonstimulated control wells. Identification of PLP antigenic determinants required that positive proliferative responses be generated to at least three adjacent overlapping 12-mers. SWXJ mice develop acute EAE ∼3 wk after conventional immunization with either of the three encephalitogenic PLP determinants p104–117, p178–191, and p139–151 . Affected mice typically undergo an incomplete recovery from the initial attack with residual neuroparalytic signs. Recovery is soon followed by a series of multiple relapse/remission cycles with each neuroparalytic episode leaving the mice progressively more impaired. By 8–12 wk after immunization, the incremental accumulation of neurologic deficit makes it increasingly more difficult to observe relapses. This chronic-progressive stage of EAE is characterized by sustained limb paresis or paralysis and marked CNS demyelination particularly pronounced in the spinal cord . To evaluate the changes occurring in self-recognition after immunization with different encephalitogenic peptides, three groups of SWXJ mice were immunized with each of the PLP determinants (p104–117, p178–191, and p139–151), and splenocytes were tested at various times thereafter for proliferative responses to the three PLP determinants as well as to the defined encephalitogens MBP 87–99 ( 20 – 22 ) and MOG 92–106 ( 23 ). Three to five independent experiments were performed for each time point. We found that the kinetics of the response to the PLP priming determinants p104–117 and p178–191 were similar, with peak responses occurring at 2–4 wk, a decline in responses by 8 wk, and a virtual absence of detectable proliferative responses to the priming immunogens by 12 wk after immunization. Although the profile of reactivity to the p139–151 priming determinant also showed a rise, peak, and decline, the kinetics of the p139–151 response were notably distinct from those of the other PLP determinants, in that proliferation took longer to peak (8 wk versus 2–4 wk) and decline (12–16 wk versus 4–8 wk) and reached the level of control responses in only two out of five mice . Nevertheless, by 12–16 wk after immunization, proliferative responses to three distinct PLP priming determinants invariably declined and were often undetectable despite the concurrent development of chronic-progressive disease . Since it was possible that the observed regression of splenocyte responsiveness to priming determinants may simply reflect a selective sequestration of autoreactive T cells from the periphery into the CNS, we simultaneously compared splenic and CNS autoreactivities during the course of disease. At distinct clinical stages of EAE, mononuclear cells were isolated from the brain and tested along with splenocytes for proliferative responses to priming determinants. In SWXJ mice immunized with both PLP 104–117 and PLP 178–191 , concurrent declines in responses to priming determinants were evident in both splenocytes and CNS-infiltrating cells during the course of disease. By the second relapse, complete regression of primary autoreactivity was clearly evident in both splenocytes and CNS-infiltrating cells despite the presence of similar numbers of infiltrating cells compared with onset and first relapse . These data indicate that regression of primary autoreactivity occurs simultaneously in both the periphery and CNS. During the regression of primary autoreactivity in EAE, responses invariably emerged to self-determinants not involved in the initial priming process. By 4 wk after immunization with PLP 104–117 , proliferative responses to PLP 139–151 (SI = 2.5) and MBP 87–99 (SI = 2.3) became apparent, peaked by 8 wk (SI = 3.9 and 3.5, respectively), and remained markedly elevated at 12 wk when acquired responses to PLP 178–191 (SI = 3.6) first became evident and responses to the p104–117 priming determinant declined toward baseline (SI = 1.6). In contrast to the plasticity observed in self-recognition, responses to the priming adjuvant Mycobacteria tuberculosis H37RA showed little fluctuation throughout the testing period (SI = 6.13 ± 0.24 SEM). Thus, a cascading emergence of neo-autoreactivity accompanied the regression of primary autoreactivity during the development of chronic-progressive disease induced by immunization with PLP 104–117. Epitope spreading also accompanied disease progression in SWXJ mice immunized with PLP 178–191 as indicated by the appearance of proliferative responses to PLP 139–151 (SI = 3.2) and MBP 87–99 (SI = 2.2) 4 wk after priming with the PLP 178–191 immunogen . Moreover, the observed neo-autoreactivity remained elevated at 12 wk (SI = 3.3 and 3.2, respectively), when proliferative responses to the priming PLP 178–191 determinant were virtually undetectable (SI = 1.4). Responses to H37RA were similar at all times tested (SI = 4.4 ± 0.21 SEM). As described in our previous report ( 6 ), a cascading epitope spreading pattern occurred after immunization of SWXJ mice with PLP 139–151. A readily detectable response to MBP 87–99 occurred at 8 wk (SI = 2.6) and remained elevated at 12 wk (SI = 2.9) when additional neo-autoreactivity to PLP 178–191 (SI = 3.6) became clearly evident . Responses to H37RA showed little fluctuation throughout the testing period (SI = 4.25 ± 0.13 SEM). To determine whether the concurrent processes of regressing primary autoreactivity and emerging epitope spreading occurs during progression of MS, a related series of experiments was performed using monocentric monophasic IMDS patients who showed no evidence of prior subclinical disease activity as determined by T2-weighted MRI. Such patients often progress to CDMS ( 8 – 10 ) and have been shown in our previous report to develop sustained autoreactivity to defined PLP peptides ( 26 ). In serial evaluation of PBMC-proliferative responses to an overlapping PLP peptide series, three IMDS patients (VS, DL, and JB) showed sustained autoreactivity to PLP determinants (p210–244, p116–150, and p117–152, respectively) coinciding with or appearing soon after the diagnosis of IMDS . Serial testing over a 3-yr period showed that in each case the established autoimmune responses associated with the early onset stage of the disease process invariably declined with time and eventually became undetectable. The two IMDS patients, VS and DL, showed progression to CDMS, and their disease progression was accompanied by the emergence of PLP neo-autoreactivity . At 154 wk after initial onset of neurologic symptoms, VS was diagnosed with CDMS after developing a cervical myelitis with a new enhancing MRI lesion of the cervical spinal cord that corresponded to new symptoms. The progression to CDMS was associated with the acquisition of newly acquired secondary responses directed against PLP 50–69 (SI = 4.7) at 154 wk and both PLP 167–185 (SI = 2.4) and PLP 258–271 (SI = 3.4) at 170 wk . At 154 wk and after, there were no detectable proliferative responses to the primary PLP 210–244 region associated with the early disease stage. Patient DL showed progression from IMDS to CDMS 60 wk after initial onset of symptoms after developing symptoms consistent with a brainstem syndrome. Disease progression was associated with the development of a secondary spreading response to PLP 49–62 (SI = 2.4) at 54 wk equal in magnitude to the autoreactivity directed against PLP 116– 150 (SI = 2.4), a determinant that once generated a vigorous sustained autoreactivity during the onset stage of IMDS . At 82 wk, the response to the spreading determinant PLP 167–182 (SI = 2.7) was greater than that elicited by PLP 116–150 (SI = 2.3) and was even greater by 162 wk (SI = 3.2) when the response to the initial autoreactive PLP 116–150 was undetectable (SI = 1.1). Thus far, patient JB has not progressed to CDMS. However, at 148 wk after initial onset of symptoms, JB developed the first indications of neo-autoreactivity with responses directed against PLP 261–274 (SI = 2.4) unaccompanied by detectable responses to PLP 117–152 associated with the early onset stage of IMDS . Throughout the experimental period, fluctuations occurring in positive control responses of VS, DL, and JB showed no correlation with changes observed in primary and spreading autoreactivities (data not shown). Our results indicate that progression of both EAE and MS is consistently accompanied by the spontaneous decrease and frequent disappearance of the established primary autoreactivity and the concurrent emergence of the epitope-spreading cascade. Our findings are consistent with the view that progression of autoimmune disease involves the sequential appearance and regression of responses to a cascading series of self-determinants so that at any given time the response to one of several distinct determinants may appear to be predominant. Indeed, based on the proliferative responses at 12–16 wk after immunization, it would be most difficult if not impossible to identify the determinant used as the priming immunogen for EAE induction in every case examined . Thus, the concept of immunodominance as it pertains to the natural development of self-recognition during autoimmune disease may best be understood when considered in the temporal context of an “epitope du jour” perspective. Our study directly challenges the widely held view that EAE and most notably MS are initiated and maintained by autoreactivity directed against a single predominant myelin protein or determinant. Several studies have claimed that autoreactivity in MS is directed in a predominant manner against specific myelin proteins and their defined immunodominant determinants. Predominant MHC class II–restricted responses have been described for peptides in the MBP 80–105 region ( 29 – 32 ) as well as for PLP peptides p40–60 ( 33 ), p89– 106 ( 34 ), p30–49, and p180–199 ( 35 ). Recent studies also claim a predominance of autoreactivity to MOG over other myelin proteins in MS patients ( 36 ). However, conclusions from these studies are based invariably on data taken at one time point from patients with long-standing disease. Thus, it is not surprising that a one-dimensional view of self-recognition would prevail from such a static perspective. Serial evaluation of self-recognition over a period of time sufficient to observe progression of clinical disease clearly reveals shifts from one predominant autoreactive pattern to another. It is likely that the level of self-recognition remodeling observed in the study reported here during progression of EAE and MS effectively under-represents the dynamic level of the changes that actually occur. This view is proposed in light of the fact that PLP served as the sole autoantigen evaluated in this study and that proliferation was the only assay used for measuring autoreactivity. The actual level of self-recognition plasticity may best be estimated by serial assessment of the autoreactive changes to overlapping peptides of several myelin proteins implicated in MS autoreactivity, such as PLP, MBP, and MOG, as well as by the incorporation of more sensitive assays such as the ELISPOT capable of providing a 10–200-fold increased sensitivity over conventional methods for detecting immunoreactivity ( 37 , 38 ) and the use of soluble peptide–MHC tetramers that enable frequency analysis of antigen-specific T cells by flow cytometry ( 39 ). It seems reasonable to speculate that shifts from one epitope response pattern to another may also be accompanied by a broadening in usage of MHC class II molecules restricting the autoreactivity. Moreover, it may be possible to sustain responses to a given determinant by expanding the autoreactive repertoire with clones that utilize restriction elements not involved in the primary response. A recent report from our laboratory showed that during progression from IMDS to CDMS, a patient homozygous for DRB1*04 responded in a DPB1*0301-restricted manner to PLP 43–64 ( 40 ), a peptide region shown in other studies to be DRB1*04 restricted ( 33 ). Thus, autoimmune spreading may involve diversification of restriction elements in addition to broadening of epitopes recognized. From this perspective, both epitope spreading and “MHC spreading” may participate together to sustain autoreactivity and thereby facilitate chronic progression of autoimmune disease. Although shifts from primary to secondary autoreactive profiles were observed in both MS and EAE, each step in the process was markedly prolonged in MS. Typically, primary autoreactivity was detectable for over 1 yr in MS compared with 12 wk for PLP 104–117 and PLP 178–191 in murine EAE. Highly developed secondary patterns of autoreactivity occurred within weeks or months of onset of clinical symptoms in murine EAE but often required years to develop during progression of IMDS to MS. However, it should be noted that the contrasting self-recognition kinetic profiles of EAE versus MS were accompanied by corresponding differences in the development and progression of clinical symptoms, i.e., progression of murine EAE occurs within a few months, whereas progression of IMDS to CDMS often develops over many months or years. Thus, it appears that the shifting autoreactivity observed in SWXJ EAE mice represents an accelerated version of the same underlying processes responsible for the development of self-recognition in MS. The shifting patterns of self-recognition shown in this study functionally reveals the fundamental underlying instability of the autoreactive T cell repertoire in MS. This view is supported by other studies showing changes in T cell repertoire restriction in IMDS patients who progress to CDMS ( 41 ). The inherent plasticity of the autoreactive repertoire has implications with regard to therapeutic applications, perhaps most notably evident in clonotypic therapies targeting specific TCR genes. Such T cell vaccination approaches result in the emergence of new autoreactive repertoires using TCR genes distinct from those used by the original responding T cells ( 42 ). Thus, the inherent capacity of the immune system to provide a continual source of neo-autoreactivity may serve ultimately as a basis for undermining the effectiveness of TCR-based therapies that fail to target the secondary spreading repertoires. Recently, it has been suggested that it may be possible to harness epitope spreading in such a manner as to facilitate the spread of immune suppression during autoimmune disease ( 43 ). Prior reports have shown that chronic progression of EAE can be inhibited by inducing intramolecular ( 5 ) or intermolecular ( 6 ) tolerance to determinants of the epitope spreading cascade. Recent studies in our laboratory indicate that adoptive Th2 immunotherapy targeting spreading determinants results in a marked long-term inhibition of EAE progression even when transfer occurs before the development of endogenous self-priming ( 44 ). Thus, stacking the T cell repertoire to favor an active Th2 response to spreading determinants may subvert the neo-autoreactive process and produce a long-lasting therapeutic outcome. In this regard, peptide-based therapies such as those incorporating the HFFK amino acid motif of the putative human immunodominant MBP determinant ( 45 , 46 ) or those involving altered peptide ligand strategies ( 47 , 48 ) may prove to be most effective if the repertoire capable of responding therapeutically to the selected peptide has undergone minimal spontaneous regression. It is evident from this study that progression of both EAE and MS may occur in the absence of primary initiating autoreactivity. However, thus far the basis for regression of primary autoreactivity is unclear. Our data indicate that the disappearance of primary self-recognition is not due to sequestration of autoreactive T cells in the CNS thereby creating an apparent loss of systemic autoreactivity. Therefore, more intricate explanations are needed to account for the spontaneous disappearance of self-recognition. Chronic self-stimulation may result in T cell exhaustion and peripheral clonal deletion ( 42 ), perhaps through apoptosis ( 49 , 50 ). Alternatively, autoreactive T cells may be present but unreactive as a result of T cell anergy ( 51 – 54 ) or suppression ( 55 , 56 ). Studies designed to determine the underlying mechanism(s) responsible for the regression of primary autoreactivity and the emergence of epitope spreading are currently in progress. | Other | biomedical | en | 0.999996 |
10190895 | Leukemic cells were isolated from bone marrow and spleen of leukemic hMRP8-PML/RARα transgenic mice (leukemia 935) as described ( 10 ), by flushing RPMI medium through long bones and collecting exudates from spleen. In vitro, spleen cells were cultured in RPMI medium supplemented with 10% FCS and 2% pockweed mitogen spleen–conditioned medium and were left untreated or were treated with 1 μM RA, 1 μM As 2 O 3 ( Sigma Chemical Co. ), or both. Leukemias were propagated by injecting blasts (10 7 viable hematopoietic cells) into the tail vein of 6–7-wk-old syngenic FVB-NICO mice. Animal handling was done according to the guidelines of institutional animal care committees. Mice implanted with leukemic cells were randomly assigned to either type of treatment. RA was administrated to leukemic mice by subcutaneous implantation of a 21-d release pellet containing 10 mg ATRA (Innovative Research of America). A stock solution of 330 mM As 2 O 3 was prepared by diluting the powder in 1 M NaOH, then a dilution in Tris-buffered saline (TBS) was administered by daily intraperitoneal injection at the concentration of 5 μg/g mice. Control mice were treated with placebo pellets or intraperitoneal injections of TBS. Specimens of spleen, liver, and lung were cut into three parts and immediately processed for snap freezing in liquid nitrogen or fixations. Specimens of long bones were fixed in formaldehyde, decalcified in 10% nitric acid, and further processed for paraffin embedding. Spleen, liver, and lung were either fixed in alcohol-formaldehyde-acetic acid reagent (AFA; Carlo Erba Laboratories), paraffin embedded and stained with hematoxylin-eosin and May-Grünwald-Giemsa, or fixed in 2.5% glutaraldehyde in cacodylate buffer and epon embedding for electron microscopic examination. The extent of the leukemic infiltrate was assessed on paraffin sections. The differentiation of the leukemic cells was assessed by combining cytological and histological stains, immunofluorescent staining of cryocut sections with a rat anti–mouse CD11b antibody ( PharMingen ) and electron microscopic analysis. In situ cell death was studied by morphological analysis on paraffin sections, electron microscopic grids, and by terminal deoxynucleotidyltransferase– mediated dUTP nick end labeling (TUNEL) assays (reagents from Boehringer Mannheim ), both on paraffin and cryocut sections. hMPR8-PML/RARα transgenic mice develop transplantable leukemias which differentiate both in vivo and in vitro upon RA exposure ( 10 ). To test their sensitivity to arsenic in vitro, leukemic cells were isolated from spleen or bone marrow of moribund animals and cultured in the presence or absence of arsenic. Little apoptosis and no differentiation were observed by TUNEL or cytological examinations. Conceivably, growth factors present in conditioned media may block apoptosis, as demonstrated in other cellular settings. However, both arsenic and RA induced PML/RARα degradation (data not shown), as shown previously in APL cell lines ( 25 , 26 ), confirming that degradation of the fusion protein does not suffice to trigger arsenic-induced apoptosis ( 30 ). Syngenic FVB mice were then injected with 10 7 leukemic cells. Transplantation was always successful, as all animals died with an intraexperimental variation of <1 wk, generally in 30–50 d. In dose–response experiments, mice were treated for 1 mo with daily injections of arsenic or TBS 4 d after leukemia engraftment. Although 1 μg/g body wt arsenic daily yielded no tumor regression upon killing, 10 μg/g led to many early deaths, presumably of toxic origin (pathological examination revealed some hepatic toxicity and widespread pulmonary edema). However, with 5 μg/g, arsenic-treated animals showed greatly reduced leukemic infiltrate of the organs analyzed. As nontransplanted mice treated with this same dose for the same length of time also showed no evidence for toxicity, a daily dose of 5 μg/g was used thereafter. Despite the much higher doses used in mice compared with humans, the circulating arsenic levels were in the range of those present in arsenic-treated APL patients (31; data not shown). In pilot survival experiments where mice were treated 4 d after transplantation for 38 d, the 10 arsenic-treated mice lived significantly longer than the 10 controls (mean: 124 ± 6 vs. 50 ± 4 d). Altogether, our results demonstrate that leukemic cells from PML/ RARα transgenic mice are arsenic sensitive in vivo. We have previously shown in cell lines that arsenic and RA appear to synergize for both differentiation and apoptosis ( 30 ), although this has been disputed ( 22 , 28 ). To test the possible synergy between these two agents in vivo, we evaluated their effects on the regression of established leukemias. Hence, for this set of experiments, leukemias were allowed to develop for 20–25 d before therapy. Leukemic mice were then randomly assigned to treatment with arsenic, RA, both, or vehicle for 4 or 8 d and killed (two mice per treatment and time point). In three different experiments, RA or arsenic treatments reduced spleen weight and liver infiltration, whereas their association completely normalized the macroscopic appearance of these organs (not shown). Microscopic examination of hematoxylin-eosin–stained sections of bone marrow, spleen, and liver from these animals confirmed this observation. In the absence of therapy, massive leukemic infiltration was evident in all three organs. In particular, the bone marrow was strictly monomorphic, consisting of promyelocyte-like cells that retained immature features such as basophilic cytoplasm . As reported previously, RA caused the rapid differentiation of leukemic cells into polymorphonuclear leukocytes. In the bone marrow, 4 d of RA treatment induced a drastic reduction of the cellular density with reappearance of some adipocytes . Nevertheless, the marrow remained monomorphic, almost exclusively composed of polymorphonuclear cells . After 8 d of RA, normal hematopoiesis was restored, with a large number of erythroblasts and a decrease in granulocytes compared with nonleukemic bone marrow . In the liver of animals treated with RA for 4 d, small remaining tumor masses consisting of maturating myeloid cells were found around vessels of the portal tracts or centrilobular veins . Leukemic infiltration of the parenchyme was dramatically reduced at day 8 (not shown). The spleen contained a large number of granulocytes at both 4 and 8 d, but the leukemic infiltrate rapidly diminished (not shown). These observations confirm previous analyses of these animals ( 10 ). 4 d after arsenic treatment, some cells with a condensed nucleus have apoptotic-like features , while partly differentiated cells with indented nuclei were also observed . At 8 d, the bone marrow remained quite monomorphic, consisting of myeloid cells with an altered chromatin clearly distinct from that of untreated blasts . In the liver of untreated animals, leukemic blasts infiltrate the parenchyme as very large perivascular masses associated with smaller aggregates of leukemic cells that obstructed sinusoids . In the leukemic blasts from the small intrasinusoid aggregates, arsenic induced morphological changes such as the appearance of indented nuclei and apoptosis-like nuclear condensation . As a result of arsenic therapy, only large perivascular masses consisting of differentiated/apoptotic cells remain after the first week (not shown). Nevertheless, at both time points, the reduction in tumor mass was less drastic than that observed with RA. Treatment with both RA and arsenic led to a much faster decrease in the leukemic population. In the marrow, islets of normal erythroblasts were already clearly visible 4 d after treatment, which was not the case with the single agent treatments . After 8 d, the bone marrow was normal, with abundant erythroblasts and megakaryocytes . Interestingly, we found numerous activated phagocytes with internalized granulocytes, which could account for the relative deficit in granulocytes compared with nonleukemic marrow. 4 d after treatment, the liver presented only very small remaining aggregates of leukemic cells around large vessels . At 8 d, both liver and spleen appeared tumor-free (not shown). Ultrastructural analysis of liver sections was undertaken to analyze the morphology of leukemic cells after 4 d of therapy . In livers of untreated leukemic animals, blasts (with lobulated nuclei and dense cytoplasm with some granulations) were clearly visible among hepatocytes and endothelial cells. Upon RA treatment, differentiating myeloid cells resembling granulocytes with fragmented nuclei and dense chromatin were found in the vascular space. Interestingly, arsenic treatment led to the appearance of many cell remnants, often consisting of naked nuclei, or with profound cytoplasmic alterations including large vacuoles and disrupted plasma membrane. However, the chromatin appeared moderately condensed at the nuclear periphery. The nuclear indentations and the presence of cytoplasmic granulations are strongly suggestive for the leukemic origin of these cells. We have recently demonstrated that PML triggers a caspase-independent cell death ( 7 ). The aspects of arsenic-treated APL blast unraveled here are highly reminiscent of PML-induced death, consistent with the idea that one of the effects of arsenic is to trigger PML- mediated death. Dual-treated specimens harbored very few hematopoietic cells, but on some occasions, images of apoptotic granulocyte phagocytosis were observed . Altogether, these analyses confirm that RA induces differentiation whereas arsenic triggers a cell death process not associated with major nuclear alterations. To quantify differentiation and apoptosis, sections were stained with CD11b for assessment of differentiation ( 22 , 29 , 30 ) and a TUNEL assay was used for assessment of apoptosis. Either RA, arsenic, or both treatments sharply induced CD11b expression in the infiltrated liver at day 4 , as shown previously for APL blasts in patients ( 29 ). In the liver, a basal level of TUNEL positivity was noted in the leukemic cells of untreated mice , consistent with high rates of spontaneous apoptosis of tumor cells in vivo. Arsenic sharply enhanced TUNEL positivity, particularly in the small leukemic aggregates in the liver sinusoids . With RA treatment, intense TUNEL positivity was found in the red pulp of spleen, whereas liver was completely negative, suggesting that RA triggered the migration of differentiated leukemic cells to the spleen where they underwent apoptosis. Double RA/arsenic therapy led to an even more dramatic enhancement of TUNEL positivity in the spleen , suggestive of accelerated differentiation and migration to this site. To see whether RA and arsenic also influenced survival, 20 mice were transplanted, allowed to engraft for 12 d, and were then left untreated or were treated with arsenic, RA, or both until the first mouse in the control group died (40 d). Hence, mice were treated for 28 d, and survival was monitored. After arsenic therapy, all animals eventually died within a narrow time range , as reported above with a shorter implantation time before treatment. In the case of RA therapy, relapses were more scattered but all animals died between 78 and 220 d after transplantation. In striking contrast, all double-treated animals were alive 9 mo after transplantation. The log–rank test demonstrates that differences between the survival of these four groups are highly statistically significant . Moreover, dual RA and arsenic therapy was significantly better than RA alone ( P = 0.002). These observations are consistent with the synergistic effects of RA and arsenic on tumor regression. To know whether the double treatment had actually eradicated the leukemia, surviving animals were killed at day 280 after transplantation. Microscopic examination of the bone marrow and spleen showed no leukemic infiltrate (not shown). The presence of leukemic cells was molecularly assessed by PCR amplification of the leukemia- specific PML/RARα fusion gene. In splenic DNA from all four mice tested, no amplification products were found with a nested PCR assay that detects 1 leukemic cell in 1,000–10,000 cells (32; data not shown), whereas the mouse p13 gene was amplified in all four cases. Thus, after dual RA and arsenic therapy, leukemic cells have become undetectable. This report presents evidence that two drugs that specifically target the PML/RARα fusion protein in APL cooperate in vivo to induce tumor regression and dramatically prolong survival. This model offers the advantage that it closely mimics the APL situation: a population of malignant cells is present in an immunocompetent organism, and only this population is PML/RARα positive, in contrast to transgenic animals where all myeloid cells express the fusion protein. The behavior of the leukemic cells versus the nontransformed hematopoiesis is much better assessed in this setting, and immune response against the leukemia can occur. Despite previous claims ( 28 ), it seems logical that these two drugs which target an oncogene for degradation through distinct pathways cooperate rather than antagonize, confirming our previous findings in vitro ( 30 ). A double dominant-negative model was proposed to explain APL pathogenesis, whereby PML/RARα blocks the functions of the normal RARα (differentiation) and the normal PML (apoptosis) proteins ( 20 ). Apart from inducing PML/RARα degradation, RA transcriptionally activates RAR, promoting differentiation. In addition, RA induces RARα degradation (30; our unpublished observations). Similarly, arsenic induces PML/RARα degradation. Arsenic also targets PML onto NBs, enhancing its proapoptotic properties ( 7 ) and subsequently promoting PML degradation ( 25 ). Hence, in this double dominant-negative model, PML/RARα degradation by one agent should favor the action of the other and vice versa . Our results, both in vitro and in vivo showing enhanced differentiation and apoptosis with dual treatments, are consistent with this model. Nevertheless, it is also possible that arsenic modifies the function of RARα, as it enhances RARα phosphorylation ( 25 ) (which was recently shown to modify its function ) and induces RARα catabolism ( 30 ). Together with PML/RARα degradation, arsenic's effects on RARα could account for the moderate differentiation induced by this agent. Moreover, the most striking synergy in the double treatments concerns differentiation, suggesting that arsenic enhances RA's effects more than the reverse. Some toxicity occurred, but under our conditions it was acceptable and never led to deaths. Arsenic alone was hepatotoxic as assessed by moderate edema and steatosis, whereas dual treatment induced some hepatocyte apoptosis suggested by dense rims of nuclear heterochromatin and nuclear condensation on electron micrographs . Some endothelial toxicity was also noted with dual treatment. However, the absence of major toxicity in a pilot case of dual treatment in a relapse APL patient (Dombret, H., and L. Degos, personal communication) suggests that toxicity is unlikely to limit the association of these two drugs. In our experimental model, mice relapse quickly after single treatment discontinuation. One obvious possibility is that our treatments were too short. Alternatively, the therapeutic route (subcutaneous for RA, intraperitoneal for arsenic), different from that used in patients (oral for RA, intravenous for arsenic), may not have been optimal. Nevertheless, in human APL, resistance to RA or arsenic as single agents is quite rapid ( 31 , 35 , 36 ). In addition, rate of spontaneous resistance to RA or arsenic of APL cell lines is also high ( 30 , 37 ). Such high intrinsic resistance of APL cells to these agents could account for the high incidence of relapses with single agent therapy. Here, the apparent eradication of the leukemic clone may reflect the direct differentiating/ proapoptotic properties of these two agents. Alternatively, the small number of cells resistant to both RA and arsenic may be eradicated by NK cell activity or by an immune response against the graft. In that sense, the necrotic-like death of arsenic-treated APL cells could induce an antileukemia immune response, as proposed in another setting ( 38 ). To our knowledge, these studies represent the first example of clinical trials in a mouse model derived from a transgenic system of a human leukemia. Current protocols use induction therapies based on the simultaneous or sequential use of RA and chemotherapy ( 39 ). To date, arsenic is used as a single agent, principally in relapse APL patients ( 31 , 35 ). The dramatic synergy between these two agents has obvious therapeutic indications: eradication of the leukemic clone in dual-treated animals clearly favors the use of arsenic as a first line drug, suggesting that combined therapies should be assessed in APL patients. | Study | biomedical | en | 0.999996 |
10190896 | BDC2.5 TCR transgenic mice have been described previously ( 32 ). B6. lpr mice were obtained from Dr. John Russell (Washington University, St. Louis, MO), who originally obtained congenic breeding pairs from The Jackson Laboratory . These mice were backcrossed >12 generations to C57Bl/6 (B6) and were maintained by brother/sister mating. IFN-γR–deficient mice ( 33 ) were obtained on a 129 background from Dr. M. Aguet (Institut de Recherche sur le Cancer, Epalinges, Switzerland). TNFR p55-deficient ( 34 ) and p75-deficient ( 35 ) mice were obtained as doubly deficient mice on a mixed 129 × B6 background from Dr. R.D. Schreiber (Washington University; with permission from Drs. W. Lesslauer, Roche, Basel, Switzerland, and M. Moore, Genentech , South San Francisco, CA). A control TNF-α wild-type line was derived from negative littermates and used as controls in all TNF-α receptor experiments. Mice deficient in iNOS ( 36 ) were obtained from Taconic Farms on a pure 129/SvEv background by permission of Dr. J. Mudgett (Merck Research, Rahway, NJ). All mice were bred and housed under specific pathogen free conditions. All donor islets were derived from the original knockout or mutant lines as indicated above, unless specifically noted in the text. Mice deficient in the p55 receptor were backcrossed onto NOD. scid for seven generations and intercrossed to generate p55-deficient NOD. scid mice. Flow cytometry was performed on a Becton Dickinson FACScan ® . We purchased PE-conjugated anti-CD4 (Caltag Labs.), anti-B220 ( PharMingen ) and goat anti–mouse IgM (Jackson ImmunoResearch Labs.). The mAb to the β chain of the transgenic TCR, anti-Vβ4 ( 37 ), was conjugated with FITC. List mode data was collected on 10 5 cells and reanalyzed on a PC using WinMDI (version 2.7) software written by J. Trotter ( http://facs.scripps.edu ). Diabetes was assessed by measurement of venous blood using a Bayer Glucometer Elite one-step blood glucose meter. Animals were considered diabetic after two consecutive measurements ≥250 mg/dl (13.75 mM). Onset of diabetes was dated from the first consecutive reading. Streptozotocin was prepared fresh for each set of injections in sodium citrate–buffered saline. NOD. scid mice were weighed and streptozotocin was injected intravenously at a dose of 180 mg/kg, after which most mice became diabetic (>400 mg/dl) within 48–72 h. Diabetic mice were transplanted with islets within 48–72 h of the onset of diabetes. Mouse islets were isolated by collagenase technique ( 38 ) and purified on Ficoll gradient. Individual clean islets were selected and cultured overnight at 24°C in 5% CO 2 in DMEM supplemented with 5% FCS (Hyclone), 10 mM Hepes, 5 × 10 −5 M 2-ME, penicillin (100 U/ml), streptomycin sulfate (100 μg/ml), and glutamine (2 mM). 250 islets from mutant or control mice (all H-2 b on either a 129 or B6 background) were transplanted under the renal capsule of streptozotocin-induced diabetic NOD. scid mice as previously described ( 39 ). In brief, under anesthesia (87 mg/kg of sodium pentobarbital), islets were transplanted under the renal capsule by exposing the left kidney through a flank incision, pushing the kidney through the incision, and holding it in place with small clamp attached to fatty tissue; with the aid of a dissecting microscope the capsule was cut with a needle and islets were then delivered through the incision by a Hamilton syringe fitted with a polyethylene catheter. After the catheter was withdrawn and the capsule was sealed by a small, pen-size eyecautery, the kidney was returned to the abdomen and the incision was closed. Normoglycemia was reestablished within 24 h of successful transplantation. Mice were then rested for 10–14 d to allow for vascularization of the graft by host vascular endothelium before the introduction of diabetic T cells. In the mixed islet grafts, the number of islets was 300 (200 p55 −/− and 100 wild-type). Transplants were performed in a similar manner on bilateral kidneys with a total of 300 islets being transplanted per mouse. In all experiments, mice not receiving diabetogenic T cells remained normoglycemic (80–110 mg/dl) for >180 d. Confirmation of the graft's support of normoglycemia was verified by nephrectomy of engrafted kidney. All transplants and manipulation of the mice were performed following written protocols that had the prior approval of the Washington University School of Medicine Animal Studies Committee. All necessary steps were taken to minimize the discomfort of the transplanted animals. Mice were anesthetized with 87 mg/kg of sodium pentobarbital and the engrafted kidney was exposed by a flank incision as above. The engrafted kidney was raised and freed from fatty tissue as before. The renal artery and vein along with the ureter were clamped off with a mosquito hemostat and were sutured distal to the kidney with 4-0 silk. The kidney was cut free with a scalpel. The clamp was released slowly and the suture was inspected for leaks and the incision closed. Kidney graft sections were stained with antibodies against Vβ4 (KT4), macrophages (ERMP-23; reference 40 ), dendritic cells (NLDC-145; Harlan sera labs), mad-CAM (MECA-367; reference 41 ), and peripheral node addressin (MECA-79; reference 41 ) as previously described ( 42 ). In vitro T cell proliferation to islet cell antigen was performed as previously described ( 32 ). In vivo T cell proliferation was performed using the cell surface dye, 5,6-carboxy-succinimidyl-fluorescein-ester (CSFE). Preparation and labeling of T cells with CSFE was performed as described in reference 43 . CSFE-labeled BDC2.5 T cells (10 7 cells) were transferred intravenously in to NOD. scid mice carrying kidney grafts of either 250 p55-deficient or 250 p55-sufficient islets. T cells from the draining lymph nodes (peri-renal) were collected on the indicated day after transfer and analyzed by flow cytometer for evidence of cell division. The ability to target loss-of-function mutations to specific organ systems remains a major challenge. With the exception of certain mutations targeted to the immune system using the Rag-mutant complementation system ( 44 ) or the inducible knockouts ( 45 ), it has been difficult to study the effects of broadly expressed or broadly acting mutations on specific organ systems or disease models. This is particularly true of the ubiquitously expressed cytokine receptors and their pluripotent ligands, the cytokine molecules themselves. Several experimental models have long suggested that cytokines influence the development of autoimmune diabetes (for review see reference 46 ). Although these models have been informative, it has remained difficult to attribute particular stage dependency to any given cytokine, especially under physiological conditions. Moreover, it has been difficult to determine what cellular conduit channels the action of each cytokine; is it through the effector lymphocyte, the APC, the vascular epithelium, or the islet tissue itself? Therefore, we undertook to develop a novel approach that compartmentalizes the action of cytokines and their receptors to specific cellular targets in an effort to establish the dependency of particular phases of diabetes development to the local effect of these mediators. We did this by creating chimeric NOD mice. Unlike prior models, these mice harbored wild-type NOD APCs that express the disease-associated MHC class II, I-A g7 , and wild-type NOD vascular endothelium. Moreover, they carried a defined population of diabetogenic CD4 + T cells (BDC2.5 TCR transgenic T cells) that respond to pancreatic β cell antigen, presented uniquely by NOD APCs, and are capable of mediating the autoimmune destruction of pancreatic β cells ( 32 , 47 – 49 ). What distinguishes our current approach was the prior replacement of the endogenous β cells with those derived from one of several mouse strains deficient in key cytokine receptor or proapoptotic effector molecules. In so doing, we created chimeric NOD mice containing altered β cell target tissue. This allowed us to assess the potential impact of each genetic alteration on the islet cell's ability to serve as a target for autoimmune-mediated destruction by altering the host effector lymphocyte, APC, or vascular endothelium. As shown schematically in Fig. 1 , NOD. scid mice were treated with streptozotocin, a β cell toxin, to destroy endogenous β cells, producing a chemically-induced diabetes (≥400 mg/dl). Normoglycemia (<100 mg/dl) was rapidly returned with the transplantation of ∼250 islets under the left kidney capsule. (It should be noted that the donor islets can be from any strain of mouse as NOD. scid do not reject allogeneic tissue. We routinely used islets of H-2 b donors.) The rescued mice were then rested for 7–10 d, allowing for host-derived vascularization of the graft. At this point, splenic T cells from diabetic BDC2.5/NOD. scid mice were transferred into these chimeric NOD. scid mice, and the mice were followed for onset of diabetes. These experiments are predicated on the following observations. First, as mentioned above, the recipient mice are scid , hence they do not reject allogeneic islet grafts, as confirmed by control experiments where each series of donor islets are engrafted under the kidney capsule and the mice are left unmanipulated for >180 d. None of these mice develop diabetes during this period. Moreover, the transplanted islets are functional, and are responsible for the maintenance of normoglycemia, as removal of the engrafted kidney results in hyperglycemia (data not shown). Second, the BDC2.5 T cells do not recognize the β cells directly but rather require the transfer of islet antigen to NOD (H-2 g7 ) APCs, which are supplied by the NOD. scid recipient mice. Third, although the recognition of antigen is MHC restricted, all strains of mice express the relevant antigen in their pancreatic β cells ( 48 ). And finally, once activated by antigen, BDC2.5/NOD. scid T cells can mediate the destruction of islet β cells in NOD. scid mice ( 6 ). Pancreatic β cell death during the course of T cell–mediated diabetes is by apoptosis ( 6 – 8 ). One potential mediator of β cell apoptosis is Fas (CD95). The engagement of Fas, a proapoptotic member of the TNFR family, on the surface of target cells by Fas ligand–expressing T lymphocytes leads to the apoptotic destruction of the Fas-expressing target cell (for review see reference 50 ). Treatment with IFN-γ induces the expression of Fas on the surface of a variety of cell types including β cells ( 24 ). Moreover, islet-infiltrating T cells can induce Fas expression on β cells through localized production of IFN-γ ( 31 ). Additionally, Fas-deficient, NOD lpr/lpr mice do not develop diabetes ( 30 , 31 ); however, these mice had substantially altered T and B cell immunity ( 30 ). These results notwithstanding, it has not been formally demonstrated that Fas expression on β cells is required for their autoimmune-mediated destruction. To test whether Fas expression on β cells is obligatory, we used our chimeric NOD mice model to create mice specifically lacking Fas expression on their islet cells. This was done by either eliminating the islet's ability to respond to IFN-γ by replacing the existing islet mass with islets lacking the IFN-γR or by using islets from B6. lpr/lpr mice that lack functional Fas expression as islet donors. After transplant, T cells from diabetic BDC2.5/NOD. scid mice were transferred into these mice and diabetes onset was followed. We found that BDC2.5 T cells destroyed B6. lpr/lpr islets as efficiently as control B6 islets, as shown in Fig. 2 a, indicating that Fas does not play an obligatory role as the critical inducer of β cell destruction, at least with respect to disease transferred by diabetogenic CD4 + T cells. Similarly, when NOD. scid mice were transplanted with islets deficient in IFN-γR, the chimeric mice developed diabetes at the same rate as control islet grafts . These results clearly demonstrate that islet cell Fas expression, either induced or constitutive, is not required for islet destruction by diabetogenic CD4 + T cells. Moreover, this would suggest that much of the protection seen in Fas-deficient NOD mice results from altered lymphoid development in the absence of Fas expression on T and B lymphocytes. Parenthetically, these results help to clarify the role IFN-γ may play in diabetes development. Wang et al., in describing the introduction of systemic IFN-γ receptor deficiency onto the NOD background, found that both NOD and BDC2.5/NOD mice lacking IFN-γR had severely retarded insulitis development ( 21 ). Our results would suggest that this is probably due to an effect IFN-γ has on the T cells, APCs, or host endothelium but not on the islet mass itself. Previous studies have indicated that IL-1 stimulates the production of nitric oxide (NO) either by priming for Fas-mediated apoptosis or by inducing the inducible form of the NO synthase gene (iNOS or NOS2; references 51 , 52 ). iNOS-mediated NO production can lead to β cell death in vitro ( 27 – 29 ). NO can be produced by the islets themselves or by the infiltrating macrophage/dendritic population. We found that the in vivo neutralization of IL-1β with a cocktail of antibodies and soluble receptor did not prevent NOD mice from becoming diabetic (data not shown), leading us to question its role in β cell death. However, to examine the effect that the targeted iNOS deficiency in islets had on β cell destruction, we tested the ability of iNOS-deficient islets to resist T cell–mediated destruction. As shown in Fig. 2 b, iNOS-deficient islets were destroyed with similar kinetics and magnitude as wild-type islets, indicating that iNOS gene expression is not critical for islet cell apoptosis. Although this result does not rule out a role for NO production by infiltrating macrophage/dendritic cells, it clearly demonstrates that islets themselves do not need to produce intracellular NO to undergo T cell–mediated elimination. TNF-α, which is secreted principally by activated macrophages and CD4 + Th1 cells ( 12 , 13 ), can both retard and exacerbate the development of IDDM in NOD mice largely dependent on the time of its administration ( 14 – 16 ). Thus, when TNF-α is given to NOD mice from birth to 3 wk of age, diabetes is accelerated, and conversely the administration of neutralizing antibody to TNF-α during this period markedly reduces both insulitis and diabetes ( 16 ). Yet when administered to adult NOD mice with established insulitis, TNF-α attenuates the disease process, and its antibody neutralization exacerbates diabetes ( 16 , 53 ). Moreover, the transgenic expression of TNF-α in the islets of adult NOD mice leads to insulitis without disease ( 17 – 19 ) and produces a state of T cell tolerance to islet cell antigens ( 19 , 20 ). Thus, although these experiments reveal the potent ability of TNF-α to alter the development of autoimmune diabetes, the physiological role played by TNF-α has yet to be fully elucidated. To investigate the role that localized production of TNF-α can have on the development of diabetes, we transplanted streptozotocin-treated NOD. scid mice with islets rendered doubly deficient for both TNF-αRs (TNF-αR1, p55; TNF-αR2, p75). As before, the transfer of diabetogenic T cells led to the rapid destruction of wild-type islet grafts ( 7 out of 8); however, doubly deficient islets (p55 −/− p75 −/− ) remained functional . Mice engrafted with p55 −/− p75 −/− islets remained normoglycemic for up to 52 d after the transfer of T cells. To confirm that the introduced islets were responsible for the maintenance of blood glucose, normoglycemic p55 −/− p75 −/− islet recipients were hemi-nephrectomized at day 28 to remove the engrafted kidney. As shown in Fig. 2 c, these mice became hyperglycemic within 24 h of nephrectomy, proving that the transplanted p55 −/− p75 −/− islets were indeed responsible for the maintenance normoglycemia. Interestingly, the mice carrying p55 −/− p75 −/− islets contained BDC2.5 T cells as measured by flow cytometric analysis of spleen and lymph node. In addition, these T cells were phenotypically normal in that they could still transfer disease to unmanipulated NOD. scid mice (data not shown). To assess which receptor conferred the protection, we produced chimeric mice carrying islets defective in either the p55 receptor or the p75 receptor. Fig. 2 c shows that p55 −/− islets were protected from T cell–mediated destruction, whereas the p75 −/− islets were efficiently destroyed. All the p75 −/− transplanted mice (11 out of 11) became diabetic by day 12, whereas the p55 −/− transplanted mice (9 out of 9) remained normoglycemic until end of the assay (≥28 d). We therefore concluded that the engagement of the p55 receptor by locally produced TNF-α was critical in the subsequent destruction of β cells. One explanation for the lack of islet destruction of the p55 −/− grafts is that the p55 −/− islets are non- or poorly antigenic. To test this, BDC2.5 T cells were cocultured with NOD APCs in the presence of dispersed p55 −/− and p75 −/− islet cells for 72 h under standard conditions, and T cell proliferation was measured. As shown in Fig. 3 , BDC2.5 T cells proliferated equally well to both receptor-deficient islet cells, indicating that islet cell antigenicity does not depend on TNF-αR expression. We performed a similar assay with intact islets in vitro in the presence and absence of exogenous recombinant TNF-α and were unable to detect a difference in the induced proliferation of BDC2.5 T cells (data not shown). We therefore concluded that at least in vitro, there is no difference in the antigenicity of p55 −/− and p75 −/− islet cells. This is somewhat discordant with recent results by Green et al., who reported that the localized production of TNF-α in β cells of transgenic NOD mice enhanced autoantigen presentation to BDC2.5 T cells in vitro ( 54 ). An alternative explanation for the p55-deficient islets' resistance to T cell–mediated destruction resides with a fundamental modification in the cellular constituency of the infiltrate. To evaluate this possibility, we performed an immunohistochemical analysis of both p55 −/− and wild-type (or p75 −/− ) islet grafts after T cell transfer. Engrafted kidneys were sampled at day 5, 7, and 9 after T cell transfer as well as at the time of diabetes or in the case of normoglycemic mice at day 28. As seen in Fig. 4 , there was no infiltration in either p55-deficient or wild-type islet grafts at day 5. At day 7, however, the wild-type graft showed distinct signs of peri-islet accumulation of leukocytes, with some grafts showing evidence of intra-islet infiltration and destruction. The same was not true for the p55-deficient islet grafts, which showed only modest peri-islet infiltration and no intra-islet infiltration. As seen in Fig. 4 , the most dramatic difference was revealed at day 9 when the wild-type islet grafts were completely infiltrated. These islets showed discrete foci of apoptotic β cells as revealed by TUNEL analysis (data not shown). In contrast, the p55-deficient islet grafts were only mildly infiltrated at day 9 and showed no signs of apoptosis (data not shown). Moreover, by day 13, the wild-type islet grafts were destroyed and the mice were overtly diabetic. Surprisingly, the mild infiltration of the p55 −/− grafts failed to progress, and in fact resolved, so that by day 28 they were nearly indistinguishable from those seen on or before day 5. We therefore concluded that the p55 −/− islet grafts could not sustain the infiltrating CD4 + T cells and that the propagation of destructive insulitis requires a TNF-α– dependent response on part of the islets. We then asked if the composition of the infiltrate was modified between the p55 −/− and wild-type lesions. We compared the cellular components of the transient infiltration of p55-deficient islet grafts at day 9 with those of the wild-type grafts. In general, the composition of the infiltrate was similar to that seen in the pancreata of NOD. scid recipients of T cells from diabetic BDC2.5 mice ( 42 ). Moreover, we saw no difference between the p55 −/− and the wild-type grafts in the activation state of the high endothelial venule (HEV) as revealed by staining for madCAM (MECA 367) and peripheral node addressin (MECA 79, data not shown). We were also able to identify the presence of roughly equal numbers of BDC2.5 T cells in both infiltrates as revealed by Vβ4 (KT4) and anti-CD4 staining. Both lesions contained similar subsets of macrophage (F4/80, MOMA 1, MOMA 2) and dendritic cells (NLDC-145) as well. Despite these similarities, there was one striking difference between the p55 −/− and wild-type grafts: the complete absence of β cell apoptosis in the p55-deficient grafts (data not shown). We therefore concluded that apart from the lack of continued progression of the lesions and the lack of β cell apoptosis, there was little difference in the nature of the infiltrates and the vascular endothelium between wild-type and p55-deficient islet grafts. To verify that the resistance to diabetes resided with the p55-deficient islets rather than with the host endothelium or APC populations, reciprocal transplants were performed in which wild-type islets (from 129 mice) were transplanted under the kidney capsule of streptozotocin-treated p55-deficient NOD. scid mice (N7 generation). Under these conditions, both the host vasculature and APC population lacked p55 receptor expression, whereas the engrafted islet mass retained full p55 functionality. When purified diabetogenic CD4 + T cells were transferred into these mice, diabetes developed with similar kinetics in both these mice and control recipients . This indicated that functional expression of the p55 receptor on the islet mass alone was sufficient to drive β cell destruction regardless of the p55 receptor expression status of the host APCs and the vascular endothelium. Although the p55-deficient islets were no less antigenic than the p55-sufficient islets and were equally capable of attracting similar subsets of infiltrating leukocytes, they were clearly incapable of providing a microenvironment that supported the maturation of the immune response to a point were β cell death could occur. This could be for one of two reasons. First, the propagation of insulitis may require TNF-α–mediated β cell death. In this case, the TNF-α produced locally by the infiltrating T cells and macrophages would fail to kill the p55-deficient β cells and insulitis would subside due to a lack of β cell damage. This would be consistent with our failure to observe β cell apoptosis in p55-deficient islets during the early phase of infiltration, yet it also seemed unlikely as ectopic expression of super-physiologic levels of TNF-α by the islets of transgenic mice did not lead to β cell death or diabetes ( 17 – 19 , 55 ). Alternatively, the evolution of insulitis from a benign accumulation of leukocytes to a destructive infiltrate may require a TNF-α–dependent change in the islet mass—either the release of an islet cell–produced chemoattractant or activation factor or an alteration in the secretion or production of antigen (something we are unable to mimic in vitro, but which has been observed by others, see reference 54 ). Either way, the net result would be the full activation of the infiltrating BDC2.5 T cell population such that it can now act to target β cells for destruction in a TNF-α–independent fashion. To distinguish between these two possibilities, we designed and produced chimeric NOD. scid mice that carried mixed grafts containing both varying amounts of p55-deficient and p55-sufficient islets. If TNF-α acted strictly as a cytolytic agent, only the p55-sufficient islets would be destroyed upon transfer of diabetogenic T cells, while the p55-deficient islets would be spared and normoglycemia would be maintained, provided that adequate amounts of p55-deficient islets were included in the mixed graft. On the other hand, if TNF-α acted to alter the local environment in favor of T cell activation, the presence of even a modicum of TNF-α–responsive islets would result in T cell activation and the destruction of both the p55-deficient and -sufficient islets and diabetes. We first ascertained the minimum number of islets required in our grafts to maintain a persistent state of normoglycemia in our Streptozotocin-treated NOD. scid mice. We found that as few as 100 islets could maintain blood glucose levels at ≤100 mg/dl (data not shown). Therefore, for our initial experiments we chose to mix ≥200 p55-deficient islets with ∼100 wild-type islets per graft. In this way, the “protected” p55-deficient islets were in sufficient excess to assure normoglycemia if all of the wild-type islets were destroyed. As before, mixed islet recipients received diabetogenic T cells 7–10 d after islet transplantation. As depicted in Fig. 5 b, both the mixed islet recipients and the control mice engrafted with 300 wild-type islets developed diabetes with comparable kinetics (between days 16 and 18 after transfer). In subsequent experiments, the numbers of wild-type islets were reduced until as few as 10 wild-type islets were mixed with ∼300 p55-deficient islets, yet the results (islet graft destruction and diabetes) were the same (data not shown). Therefore, we concluded from these experiments that the stimulation of islet cells through their p55 receptor altered the local environmental conditions favoring the development of a productive BDC2.5 T cell infiltrate. Having determined that a small number of islet cells can, in response to locally produced TNF-α, support the transition from benign to destructive insulitis, it was now critical to determine if this was a purely localized effect. To address this, we performed kidney grafts on streptozotocin-treated NOD. scid mice in which wild-type islets (100 islets) were engrafted under the right kidney capsule and p55-deficient islets (250 islets) were engrafted under the left kidney capsule of the same animal. By physically separating the grafts we sought to minimize the effects between the wild-type and p55-deficient graft. If, upon transfer of diabetogenic T cells, the p55-deficient graft survived in these mice, despite the destruction of the wild-type islet grafts, then the original destruction of the p55-deficient islets in the mixed islet grafts described above resulted merely by virtue of their intimate proximity to the wild-type islets. On the other hand, if the distant p55-deficient grafts were likewise destroyed, it is more likely that the transferred T cells were altered by an encounter with wild-type islet cells. We found that the twin-kidney engrafted NOD. scid recipient mice did, in fact, become diabetic 12–16 d after receiving BDC2.5 T cells, at a rate coincidental with the development of diabetes in mice harboring dual wild-type grafts . As before, those mice engrafted with only p55-deficient islets did not develop disease. Additionally, histological analysis of the p55-deficient bilateral graft showed signs of β cell apoptosis within 1 d of the onset of destructive infiltration of the p55-sufficient graft . The ability of wild-type grafts to influence the outcome of the contralateral p55-deficient grafts indicated that TNF-α responsiveness on the part of the wild-type islets led to the activation of the transferred BDC2.5 T cells such that they were now capable of homing to and destroying the p55-deficient graft on the opposite kidney. These results led us to assess the in vivo proliferative status of BDC2.5 T cells after transfer. We reasoned that the lack of progression in the p55-deficient islet engrafted mice may be due to the inability of the p55 −/− islets to fully activate the transferred BDC2.5 T cells. In the mixed and twin-kidney graft experiments, the wild-type islets would provide an environment capable of furnishing this activation and therefore of leading to the subsequent destruction of the p55 −/− islets in a TNF-α–independent fashion. If this were true, T cell proliferation in vivo might differ between mice harboring only p55 −/− islets and those carrying only wild-type islets. This was tested by monitoring the degree of in vivo proliferation of the BDC2.5 T cells in the efferent lymph of mice harboring one or the other islet grafts under the left kidney using the decay of the integral membrane dye, CSFE, as an indicator of cell division ( 43 , 56 , 57 ). As depicted in Fig. 7 , draining lymph nodes from mice engrafted with p55 −/− islets were devoid of reactivated BDC2.5 T cells, whereas the renal lymph nodes from mice engrafted with wild-type islets contained T cells that clearly had undergone several rounds of replication. Therefore, we concluded that the most likely explanation for the infiltration and subsequent destruction of p55 −/− islets in both the mixed and twin-kidney grafts was due in part to the activation of the BDC2.5 T cells in response to wild-type islets either proximal or distal to the p55 −/− islets. This process required a TNF-α response on the part of the target islet cells but the subsequent destruction of the islet tissue was TNF-α–independent. The nature of the TNF-α response on the part of islets remains unknown, but could be an increase in antigen delivery either in direct response to TNF-α or as a result of islet cell death. In either case, this leads to the subsequent activation of our infiltrating islet-reactive BDC2.5 T cells, which then act to mediate the destruction of islet β cells in a process that does not require TNF-α. Using the same BDC2.5 TCR transgenic model, André et al. have proposed two checkpoints in the progression of diabetes, the first the formation of a benign infiltrate and the second the transition to destructive insulitis ( 58 ). We would propose that the transition through checkpoint two is dependent on the active response of islets to TNF-α. Moreover, our results are consistent with the hypothesis that early and local production of TNF-α in the islet acts to enhance the islet's antigenicity and the subsequent activation of disease-causing lymphocytes ( 16 , 17 , 53 ). In conclusion, these data, taken together, demonstrate that Fas, IFN-γ, and iNOS do not play an obligatory role in the apoptotic destruction of pancreatic β cells induced by a diabetogenic CD4 + T cell population, but that TNF-α plays a critical role in the transformation of islet-reactive CD4 + T cells from a benign state of β cell indifference to an activated state of β cell reactivity. Moreover, these results suggest, for the first time, that the islet cells themselves play an active and TNF-dependent role in facilitating their own death by providing an environment capable of perpetuating T cell–mediated insulitis. | Study | biomedical | en | 0.999998 |
10190897 | A 72-yr-old man visited our hospital with complaints of low grade fever and systemic lymphadenopathy in April 1992. His white blood cell count was 1.2 × 10 10 /liter, with 8% abnormal lymphocytes. His lactic dehydrogenase (LDH) level and serum calcium level were 4,400 IU/liter and 11 mg/dl, respectively. The diagnosis of acute type ATL was made hematocytologically and confirmed by the demonstration of monoclonal integration of HTLV-I proviral DNA in the peripheral blood and lymph node mononuclear cells. The patient was administered 10 cycles of combination chemotherapy and discharged with a partial response. However, only 3 mo after the discontinuation of chemotherapy, the patient was readmitted to our hospital with massive ascites and an elevated LDH level . Although combination chemotherapy was started again, the effect was transient and the patient died of a systemic invasion of ATL cells 3 mo after his second hospital admission. Peripheral blood was drawn several times during the patient's hospital stays. A lymph node was biopsied during his first admission, and ascites were obtained during his second admission. All materials were obtained after informed consent. Mononuclear cells were separated from the samples by centrifugation over a Ficoll gradient and cryopreserved until use. The ATL cell line KOB was established from the patient's ascitic ATL cells. The other three ATL cell lines (KK1, ST1, and SO4) used for the present study were also of primary ATL cell origin established from the respective ATL patients ( 29 , 30 ). All ATL cell lines were dependent on exogenously added IL-2 and were maintained in RPMI 1640 supplemented with 10% FBS and 0.5 U/ml IL-2. Membrane Fas was examined by flow cytometry as described previously using a Cytron flow cytometer (Ortho; 31). The resulting histograms correspond to the cell number (y-axis) versus fluorescence intensity (x-axis) plotted on a logarithmic scale. Total RNA was prepared from the cell lines and clinical samples using ISOGEN (Wako). After the removal of DNA contamination using a MassageClean™ kit (GenHunter Corp.), cDNA was made from 1 μg of total RNA using an RNA PCR core kit ( Perkin Elmer ) according to the manufacturer's protocol. Oligo dT primers were used to prime the first strand synthesis for each of the reactions. Four oligonucleotide primer pairs were designed to amplify the full length coding region of Fas according to the method of Cheng et al. ( 3 ). The reaction conditions were 35 cycles of 94°C for 70 s, 60°C for 30 s, and 72°C for 60 s (for full length Fas) or 30 cycles of 95°C for 60 s, 54°C for 60 s, and 72°C for 60 s (for Fas I, Fas II, and Fas III; Table I ). These amplified products were electrophoretically separated and visualized on an ethidium bromide–stained 2% agarose gel or a silver-stained 12.5% acrylamide gel using a GenePhor electrophoresis unit ( Pharmacia Biotech ). We cloned the RT-PCR products of full length Fas and Fas III derived from KOB or clinical samples into the pCR™II vector using a TA cloning kit (Invitrogen Corp.). All sequencing experiments were performed using an AutoRead™ sequencing kit designed for an ALF™ DNA sequencer ( Pharmacia Biotech ). The truncated full length Fas cDNA of KOB origin was inserted into the retroviral vector LXSN. This vector contains Moloney murine sarcoma virus–derived long terminal repeats and a neomycin resistance gene under the control of simian virus 40 early promoter. The recombinant retroviral vector LdelSN was purified using an EndoFree Plasmid Maxiprep kit (Qiagen) and was transfected into the ecotropic packaging cell line ψ2 using FuGENE™6 transfection reagent ( Boehringer Mannheim ). After a 48-h culture in DMEM, the transient retroviral supernatant was used to transduce the amphotropic packaging cell line PA317. The PA317 cells infected with recombinant retroviruses were cultured in DMEM for 48 h and selected with 0.8 mg/ml G-418. Supernatants of PA317 cells producing amphotropic retroviruses were used to transduce Jurkat cells. The transduced Jurkat cells were selected in RPMI 1640 containing 1 mg/ml G-418 and were cloned by the colony formation method on methylcellulose-containing culture plates. The integration site(s) of HTLV-I proviral DNA and TCR gene rearrangement were examined with a Southern blot hybridization assay using probes for the pX region of HTLV-I and TCR Cβ1 according to the described method ( 24 , 32 ). In brief, the high molecular DNA samples (10 μg) extracted from the cell lines and clinical samples were digested with restriction enzymes and size fractionated on 0.7% agarose gels. They were then denatured and transferred onto positively charged nitrocellulose membranes ( Boehringer Mannheim ) and hybridized to digoxigenin-labeled probes. Thereafter, the blots were washed at appropriate stringency and treated with an anti-digoxigenin antibody conjugated with alkaline phosphatase ( Boehringer Mannheim ), and the alkaline phosphatase was changed to a light signal by CDP-Star™ ( Boehringer Mannheim ) and exposed to film. The restriction enzymes used were EcoRI and PstI for HTLV-I proviral DNA and EcoRV and BamHI for TCR β chain gene rearrangement. In the process of early apoptosis, cells express phosphatidylserine on the outer leaflet of the cell surface membrane and consequently bind annexin V. Cell lines cultured with or without IgM anti-Fas mAb at a final concentration of 1 μg/ml were examined for annexin V binding by flow cytometry at several time points after culture using an annexin V–FITC kit (Takara Shuzo Co.) according to the manufacturer's protocol. A DNA fragmentation analysis was also performed using the same samples and an apoptosis ladder detection kit (Wako). First, 10 5 cells/100 μl RPMI 1640 supplemented with 10% FCS were cultured with or without 1 μg/ml of IgM anti-Fas mAb in 96-well tissue culture plates for 7, 16, 24, or 32 h, and proliferation status was estimated by measuring the conversion of MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) into water-soluble formazan (CellTiter 96TMAQueous; Promega ) according to the manufacturer's protocol. All experiments were performed in triplicate. Four ATL cell lines were analyzed for the expression of Fas antigen (Apo-1/CD95) by flow cytometry using an IgG anti-Fas mAb. As shown in Fig. 1 , all four ATL cell lines (KK1, KOB, ST1, and SO4) and Jurkat cells expressed Fas antigen, whereas the cell line K562 used as a negative control did not. These cell lines were cultured with 1 μg/ml IgM anti-Fas mAb, and cell proliferation status was examined. The results are expressed as a value against the control cells cultured without mAb. As shown in Fig. 2 A, after a 24-h culture with the mAb, the proliferation of KK1, SO4, and ST1 cells was significantly suppressed: to 16.8, 7.7, and 10.8% of the control level, respectively. In contrast, the KOB and K562 cells were not affected. To confirm that suppression was due to apoptosis, we performed a flow cytometric analysis of annexin V binding and a DNA fragmentation assay for the early and late phases of apoptosis, respectively. In Jurkat cells, an annexin V–binding cell population appeared 3 h after treatment with IgM anti-Fas mAb (14%), and the population was further increased at 5 h (50.1%). In contrast, no annexin V–binding cell population appeared in KOB cells . Likewise, the other ATL cell lines (KK1, SO4, and ST1) exhibited annexin V binding but K562 cells did not (data not shown). Supporting the results of annexin V binding, DNA ladder formation appeared in the three ATL cell lines (KK1, SO4, and ST1) but not in KOB and K562 cells after 3 h of culture (data not shown). These results prompted us to examine the mechanism of Fas resistance in KOB cells. To investigate the Fas mRNA, four primer pairs were designed to cover its full length coding region. These PCR reactions were named Fas I, Fas II, and Fas III (Table I ). The expected size of the bands in Fas I and Fas III was 336 and 344 bp, respectively. Fas II was expected to show two bands (339 and 276 bp) generated by alternative splicing; the larger PCR product corresponds to the membrane form, and the smaller product corresponds to the soluble form of Fas mRNA, which is 63 bp shorter than the membrane form. Irregardless of the samples, only the expected bands were observed in the Fas I and Fas II reactions. In contrast, in the Fas III reaction of KOB cells, a distinct smaller product was observed in addition to the expected 344-bp band . The sequencing analysis of full length RT-PCR products of KOB cells showed two transcripts, the normal form and a truncated form. The truncated transcript lacked 20 bp (from nucleotide [nt] 686 to nt 705, nucleotide numbers that correspond to those reported by Itoh et al. ) at exon 9 within the cytoplasmic death-signaling domain. The frame shift caused by the deletion generated a premature stop codon at amino acid 239, resulting in the production of aberrant Fas antigen which lacked the entire death-signaling domain . Hematocytologically, characteristic ATL cells with deeply indented or lobulated nuclei and relatively scanty basophilic cytoplasm were observed in the peripheral blood, lymph node, and ascites of our patient. We examined the integration pattern of HTLV-I proviral DNA and TCR β chain gene rearrangement profiles by Southern blot analysis to determine the origin of KOB cells. As shown in Fig. 4 A, one monoclonal band was observed at the same position in all primary samples when cellular DNAs was digested with EcoRI or PstI. In addition, multiple bands other than the original band of the primary samples were observed in KOB cells, suggesting the superinfection of HTLV-I in vitro. In the TCR β chain gene rearrangement profile, the same rearranged band was observed in all samples including KOB when the cellular DNA was digested with BamHI. The loss of the germline band at 12 kb was observed in all but one sample (peripheral blood) when the cellular DNA was digested with EcoRV, indicating the deletion of the TCR Cβ 1 locus in this clone . The germline band at 12 kb observed in peripheral blood indicates the presence of normal non-T cells. These results suggest that KOB cells originated from the primary ATL cells. We therefore investigated whether the primary samples also expressed the truncated Fas gene. We performed an RT-PCR analysis of Fas III in all primary samples. Interestingly, although the same smaller product observed in KOB cells was detected in peripheral blood ATL cells and ascitic ATL cells, no such product was detected in lymph node ATL cells . The sequence analysis of these products disclosed that the clinical samples from peripheral blood and ascites had the same Fas gene mutation observed in KOB cells. No such mutation was observed in the lymph node cells (data not shown). To confirm that the mutant Fas gene is responsible for Fas resistance in KOB cells, we constructed the retroviral vector LdelSN containing the truncated Fas cDNA and transferred it into Jurkat cells. The successful transfer of the gene in G418-resistant cells was confirmed by RT-PCR of Fas III and neomycin resistance gene–specific sequences and by sequencing analysis. RT-PCR analysis of Fas III showed two transcripts in all LdelSN-transduced Jurkat cell clones (RJ-9, RJ-11, and RJ-14) but only normal transcript in wild-type and LXSN-transduced Jurkat cell clones . We performed a sequencing analysis of the RT-PCR products from transduced Jurkat cells and verified the expression of transferred Fas gene (data not shown). The LXSN- and LdelSN-transduced Jurkat cells were assayed for Fas sensitivity. The flow cytometric analysis of annexin V binding showed that the LXSN-transduced Jurkat cell clone LX-2 demonstrated a marked increase of the annexin V–binding cell population of 3.3% at 0 h to 56.8% at 3 h after treatment with IgM anti-Fas mAb, and the population increased further to 77.2% at 5 h. In contrast, the LdelSN-transduced Jurkat cell clone RJ-14 showed no such increase . Using CellTiter 96TMAQueous, the proliferation status was evaluated at several time points of culture with 1 μg/ml of IgM anti-Fas mAb. The proliferation of the LXSN-transduced Jurkat cell clones LX-1 and LX-3 was significantly suppressed to 37.6 and 33.4% of the control level, respectively, but the LdelSN-transduced Jurkat cell clones RJ-9, RJ-11, and RJ-14 were not affected . The characteristic features of apoptosis, i.e., reduction of cell size, condensation and aggregation of nuclear chromatin, and nuclear fractionation were observed in wild-type Jurkat cells and LXSN-transduced Jurkat cell clones but not in LdelSN-transduced Jurkat cell clones . Most ATL cells express abundant Fas antigen on their surfaces and show apoptosis in a short term culture with IgM anti-Fas mAb ( 22 , 23 , 25 , 33 ). We reported that ATL cells from all but 2 of 33 patients expressed Fas antigen, and the Fas − ATL cells were resistant to apoptosis ( 34 ). Differences in Fas expression seem to be correlated with subtypes of ATL; the level of Fas expression was higher in chronic type than in acute type ATL patients and was inversely correlated with serum LDH activity ( 24 , 25 ). These results may indicate that the Fas/Fas ligand system plays a critical role in elimination of ATL cells. Fas gene mutations were recently reported in some malignancies. 7 cases of malignancy with Fas gene mutations were detected in 48 multiple myeloma patients by the RT-PCR method and a single-stranded conformation polymorphism (SSCP) analysis. All of these mutations were point mutations in the cytoplasmic region, and two of them were silent mutations ( 26 ). 2 cases of Fas gene mutations were found among 81 childhood T-lineage acute lymphoblastic leukemia patients using PCR and single-stranded conformation polymorphism analysis as the screening methods; one case was a heterozygous point mutation in exon 3, and another case was a homozygous alteration in the promoter region ( 27 ). The 3 cases of Fas gene mutations detected among 47 ATL patients by an RNase protection assay were a 5-bp deletion in exon 2, a 1-bp insertion in exon 2, and a loss of exon 4. Two silent point mutations were also observed ( 28 ). Rearrangement or allelic loss of Fas gene were also reported in 5 of 70 patients with non-Hodgkin's lymphoma: 3 cases of allelic loss and 2 cases of rearrangement ( 35 ). According to these reports, it is likely that Fas gene mutations, including silent mutations, occur at relatively low but diverse frequencies, ∼2.5–14.6%, and are consistently detected in lymphoid malignancies, suggesting an association between Fas mutation and disease progression in lymphoid malignancies. However, a functional analysis of the aberrant Fas was not performed in any of the above studies. In many receptor and ligand systems such as epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor β, dimerizations of receptor subunits appear to be essential for signal transduction. The truncated forms of these receptors suppress the response of wild-type receptor by heterodimerization ( 36 – 41 ). Analysis of the crystal structure of TNF receptor–TNF-β complex demonstrated that three TNF receptor molecules symmetrically bound to TNF-β trimer ( 42 ). The human soluble Fas ligand also forms homotrimers ( 43 ). It has been reported that the trimerization of the death-signaling domain within the cytoplasmic region of Fas is essential for signal transduction in the process of Fas-mediated apoptosis ( 44 ). Therefore, it is likely that KOB cells prevented apoptosis by an expression of function-ablating Fas antigen on their surfaces similar to the expression on lymphocytes of ALPS patients. The truncated Fas antigen, which lacks the entire death-signaling domain, may behave as a decoy receptor and interfere with the trimerization of normal Fas in a competitive manner, resulting in the impairment of signal transduction. To test this hypothesis, we constructed the retroviral vector LdelSN containing the truncated Fas cDNA and transferred it into Jurkat cells, which are sensitive to IgM anti-Fas mAb. The resulting Jurkat cells became almost completely resistant to IgM anti-Fas mAb, suggesting that heterotrimers between the truncated Fas and normal Fas, which was endogenously expressed in Jurkat cells, did not transduce the apoptotic signal. Although the pathological role of aberrant Fas antigen has been demonstrated in ALPS by the method of transient gene transfer, no such trial has been performed in neoplasms. The present study is, to our knowledge, the first that has successfully demonstrated the dominant negative inhibition of apoptosis by a stably transfected cell line. More interestingly, the mutated Fas gene observed in KOB cells was not detected in lymph node ATL cells, although a Southern blot analysis indicated that all of the primary ATL cells and KOB cells were of the same cell origin. These results indicate that ATL cells developed in the lymph nodes have acquired Fas gene mutation, and the subclone with the mutation spread to the whole body, escaping elimination by the Fas/Fas ligand system. It has been suspected that HTLV-I–infected T lymphocytes proliferate in lymph nodes at an early phase of malignant alteration and gradually progress from the carrier type to the smoldering, chronic, and acute types of ATL. Based on a statistical analysis of the age distribution of ATL patients, the lymphomagenesis of ATL was proposed to consist of at least five steps ( 45 ). Although abnormalities in tumor suppressor genes such as p53, p15, or p16 are supposed to be one of the steps for progression of ATL ( 46 – 48 ), the precise mechanism of lymphomagenesis remains unknown. Function-ablating Fas mutations might be involved in the multistep lymphomagenesis of ATL. It is, however, unlikely that the Fas gene mutation alone leads to a malignant transformation. Fas-deficient lpr mice do not develop malignancies, and patients with ALPS have rarely developed malignancies. Interestingly, lpr mice, which constitutively express the L-myc transgene in the lymphoid lineage, show an accelerated formation of T and B cell lymphomas, but the L-myc transgene alone did not produce such an acceleration in normal mice ( 49 ). It has been reported that mice deficient in both T cells and Fas gene develop B cell lymphoma ( 50 ). These reports suggest that lymphomagenesis requires other events besides the loss of Fas receptor function. In the present investigation, it seems feasible that lymph node ATL cells with some genetic abnormalities that are crucial for the development of ATL progressed to a more aggressive form by acquiring a Fas gene mutation. In conclusion, we found a function-ablating Fas gene mutation in an ATL cell line (KOB) and its primary ATL cells. By retrovirus-mediated gene transfer, we verified that the aberrant Fas interfered with apoptosis by a dominant negative mechanism. The absence of the Fas gene mutation in lymph node ATL cells suggests that this mutation is a late event that occurs after the development of ATL and that the ATL subclone with Fas mutation escaped from the apoptosis mediated by the Fas/Fas ligand system and spread to the entire body of the patient. Abnormality of Fas function may be one of the important steps in the progression of ATL. | Clinical case | clinical | en | 0.999997 |
10190898 | C57BL/6 (H-2 b ) wild-type, C57BL/6 IL-4 −/− , and BALB/c (H-2 d ) wild-type mice were obtained from the Department of Comparative Medicine, Stanford University breeding facility. C57BL/6 IL-4 −/− mice have been described in detail previously ( 28 , 29 ). Only male mice were used at 8–12 wk of age. Care of all experimental animals was in accordance with institutional guidelines. Host mice were given 800 cGy whole body irradiation from a 250 kV x-ray source and injected via the tail vein within 12 h. Survival and appearance of mice was monitored daily, and body weight was measured weekly. Mean body weights of surviving mice in each group were determined at day 100. Chimerism in the peripheral blood of hosts at day 100 was measured by staining PBMC from Ficoll-hypaque gradients with fluorochrome-conjugated anti–H-2 b mAbs ( PharMingen ) and analysis by one-color flow cytometry. Bone marrow cells were obtained from the femur and tibia and stained with mAbs as described previously ( 21 , 30 ). Stainings were performed in the presence of anti-CD16/32 (2.4G2; PharMingen ) at saturation to block FcRII/III receptors, and propidium iodide ( Sigma Chemical Co. ) was added to staining reagents to exclude dead cells. Three-color FACS ® analysis was performed using a modified dual laser FACS Vantage™ ( Becton Dickinson ), and data was analyzed using FACS ® /Desk software ( Becton Dickinson ; 21, 30). The following conjugated antibodies were used for staining: FITC–anti-CD8 (CT-CD8α) purchased from Caltag, Inc., and allophycocyanin–anti-TCRαβ (H57-597), PE–anti-NK1.1 (PK136), and FITC–anti-CD4 (RM4-5) purchased from PharMingen . PBMC or marrow cells from wild-type C57BL/6 mice were stained with anti-CD4, anti-CD8, and anti-TCRαβ mAbs and sorted CD4 + and CD8 + α/β + T cells (CD4 + /CD8 + ) from the blood or stringently T cell–depleted (TCD) 1 marrow cells (CD4 − CD8 − α/β − ) were obtained by flow cytometry using a FACStar™ ( Becton Dickinson ) as described previously ( 21 , 30 ). Sorted CD4 + /CD8 + T cells from the marrow were obtained by flow cytometry after enrichment of bone marrow T cells on immunomagnetic bead columns (Miltenyi Biotec). Marrow cells were first incubated with biotinylated anti–Thy-1 mAb (Caltag, Inc.) and then incubated with streptavidin magnetic beads. Thy-1 + cells were positively selected by retention on the magnetic columns and subsequent release. Sorted T cells (10 5 ) from the peripheral blood or bone marrow from wild-type or IL-4 −/− mice were stimulated in vitro with 20 ng/ml PMA ( Sigma Chemical Co. ) and 1 μM Ionomycin ( Calbiochem Corp. ) in 10% FBS and RPMI complete medium in 96-well round-bottom plates and harvested at the peak time point (48 h) as described previously ( 21 ). Supernatants were assayed for the secretion of IFN-γ and IL-4 using commercial ELISA kits ( Biosource International ). Assays were developed with avidin-peroxidase and substrate, and plates were read at 450 nm using a microplate reader ( 21 ). Histopathological specimens from the skin and large intestines of hosts were obtained 40–60 d after transplantation and fixed in formalin before embedding in paraffin blocks. Tissue sections were stained with hematoxylin and eosin and examined at 400×. To test the ability of T cells from murine peripheral blood to cause GVHD, we stained PBMC from C57BL/6 mice and isolated CD4 + and CD8 + T cells as a single pool by flow cytometry (hereafter referred to as CD4 + /CD8 + T cells). Graded numbers of these purified T cells were mixed with a constant number of unfractionated (1.5 × 10 6 ) C57BL/6 bone marrow cells and injected intravenously into lethally irradiated BALB/c mice. Injection of complete H-2–mismatched unfractionated bone marrow from C57BL/6 mice into lethally irradiated BALB/c mice allowed 100% of mice to survive for at least 100 d . Control irradiated hosts given no donor cells all died by day 14, whereas those reconstituted with the unfractionated bone marrow cells alone were complete chimeras with donor-origin (H-2 b ) blood mononuclear cells at day 100, based on staining for surface H-2 b expression and flow cytometric analysis (data not shown). The addition of graded numbers of purified CD4 + /CD8 + peripheral blood T cells to the transplant resulted in dose-dependent mortality, with the addition of as few as 1.6 × 10 4 of these cells causing a significant decrease ( P < 0.05 by the log rank test) in survival compared to transplantation of unfractionated bone marrow alone . Because ∼2% of nucleated bone marrow cells were brightly staining α/β + T cells , we next determined whether stringent depletion of T cells prior to transplant would further decrease the mortality from the addition of peripheral blood CD4 + /CD8 + T cells. Surprisingly, T cell depletion of the bone marrow actually increased rather than decreased mortality compared to transplantation with unfractionated bone marrow, and this effect was observed over a large range of doses of peripheral blood CD4 + /CD8 + T cells . Strikingly, the addition of as few as 8 × 10 3 CD4 + /CD8 + T cells from the peripheral blood to TCD bone marrow cells resulted in significantly decreased host survival as compared to either transplantation of TCD bone marrow cells alone or to hosts given 8 × 10 3 CD4 + /CD8 + blood T cells plus unfractionated bone marrow cells . Only ∼30% of the hosts given the combination with TCD marrow cells survived more than 100 d, and those that died showed typical signs of GVHD including weight loss, hunched back, ruffled fur, diarrhea, and facial swelling. Necropsies of moribund hosts with severe clinical signs after day 40 showed typical GVHD histopathological lesions of the large intestines and skin (see below). Increasing the dose of CD4 + /CD8 + peripheral blood T cells accelerated the rapidity of death of the hosts until a plateau was reached between 6.4 × 10 4 and 2.56 × 10 5 T cells. Hosts given the latter cell doses all died by day 14 after cell transplantation . To test the notion that α/β + T cells in the bone marrow were responsible for protection from GVHD, these cells were purified from the bone marrow and added back to the TCD bone marrow with 6.4 × 10 4 peripheral blood CD4 + /CD8 + T cells, a dose that resulted in 100% mortality by day 14 posttransplantation . Under these conditions, the inclusion of 2.56 × 10 5 α/β + T cells significantly delayed mortality ( P < 0.05), and the inclusion of 1.024 × 10 6 of these cells resulted in the long-term survival of 50% of the transplant recipients . This inhibitory effect on GVHD was specific for α/β + T cells of the bone marrow, in that the addition of 1.024 × 10 6 TCD bone marrow cells provided no protection from mortality . Because bone marrow α/β + T cells enriched on immunomagnetic bead columns include populations that express CD4 or CD8 markers or neither marker , we next directly assessed the capacity of purified CD4 + /CD8 + α/β + T cells from the enriched bone marrow to mediate GVHD. When these were added at a ratio of 1.28 × 10 5 or 2.56 × 10 5 donor T cells/1.5 × 10 6 TCD marrow cells, 100 and 90% of the hosts, respectively, survived for more than 100 d . The surviving hosts did not show signs of GVHD, and their mean body weight at day 100 posttransplantation did not differ significantly from that of mice that had received TCD bone marrow alone (data not shown). Bone marrow CD4 + /CD8 + T cells were therefore strikingly ineffective in inducing lethal GVHD. In control experiments, PBMC were applied to immunomagnetic bead columns, and sorted CD4 + /CD8 + T cells were obtained thereafter by flow cytometry. These T cells (3.2 or 6.4 × 10 4 ) were injected with TCD marrow cells into groups of 10 BALB/c hosts, and survival was compared to that of hosts given the same number of sorted peripheral blood CD4 + /CD8 + T cells without column enrichment. The column-enriched cells remained as effective as an equivalent number of these T cells obtained without column enrichment in mediating lethal GVHD (data not shown). Previous studies have shown that bone marrow α/β + T cells from C57BL/6 mice, as well as several other strains, have a substantially greater proportion of cells with surface expression of NK1.1 than peripheral blood α/β + T cells ( 18 , 21 ). As there is recent evidence that NK1.1 + α/β + T cells may act as negative regulators of T cell–dependent autoimmune diseases (22– 24), it was plausible that a bone marrow NK1.1 + T cell population might act in a similar negative regulatory fashion in GVHD. Therefore, we analyzed bone marrow and peripheral blood α/β + T cells for their surface expression of CD4 plus CD8 versus NK1.1. This revealed that ∼30% of C57BL/6 PBMC were α/β + T cells that expressed either CD4 or CD8 . CD4 and CD8 are expressed in a mutually exclusive pattern ( 21 ). Less than 1% of the peripheral blood α/β + T cells expressed NK1.1 , in agreement with previous reports ( 19 , 20 ). In contrast, ∼20% of bone marrow α/β + T cells were CD4 − CD8 − , and most of the CD4 − CD8 − T cells were NK1.1 + . 16% of bone marrow α/β + T cells were NK1.1 + and either CD4 + and/or CD8 + (upper box). Further analysis of gated α/β + T cells from the bone marrow showed that the percentage of NK1.1 + CD8 + T cells exceeded that of NK1.1 + CD4 + T cells . Although NK1.1 + CD8 + T cells were easily identified in the bone marrow, this subset was not detected in the peripheral blood, thymi, or spleens of the donor mice (data not shown). Because the CD4 + /CD8 + T cell subset of bone marrow cells contained a substantial proportion of NK1.1 + cells but the peripheral blood did not, we tested the ability of purified CD4 + /CD8 + T cells depleted of NK1.1 + T cells to induce GVHD. As before, the addition of 2.00 × 10 5 CD4 + /CD8 + T cells (including the NK1.1 + population) to the TCD bone marrow transplant induced minimal clinical signs of GVHD in irradiated BALB/c hosts, and no deaths occurred during a 100-d observation period . In contrast, an equal number of NK1.1-depleted CD4 + / CD8 + T cells added to TCD bone marrow resulted in the death of ∼70% of hosts within 20 d and was associated with clinical signs of GVHD including facial swelling, hair loss, hunched back, and weight loss . Reduction of the NK1.1-depleted CD4 + /CD8 + T cell dose to 1.00 × 10 5 cells still resulted in the death of ∼40% of hosts by day 20. This was a significant reduction in survival ( P < 0.01, log rank test) as compared to the group given sorted CD4 + / CD8 + T cells. Histopathological examination of the large intestines and skin of additional mice given 2.00 × 10 5 NK1.1 − T cells showed lesions of GVHD, including inflammation in the intestinal crypts and hyperplasia and apoptosis of crypt epithelial cells, inflammation of the dermis, and epidermal hyperplasia as compared to mice given TCD bone marrow alone . Unlike conventional α/β + T cells, NK1.1 + T-lineage cells of the thymus and spleen rapidly secrete large amounts of IL-4 and IFN-γ after engagement of the TCR–CD3 complex or after stimulation with calcium ionophore and phorbol PMA, without previous exposure to antigens or polyclonal T cell activators ( 31 – 33 ). IFN-γ and IL-4 are important in helping to polarize T cell responses toward Th-1 or Th-2 patterns of cytokine production that have been implicated in the induction and amelioration of GVHD, respectively ( 34 – 36 ). Therefore, we characterized the capacity of bone marrow T cells, including the NK1.1 + population, to produce these cytokines after in vitro stimulation with calcium ionophore and PMA for 48 h, a time point at which cytokine levels were maximal ( 21 ). Depletion of NK1.1 + T cells from the bone marrow CD4 + /CD8 + T cell population resulted in an eightfold reduction of IL-4 production (Table I ) that was highly significant ( P < 0.01, two-tailed Student's t test). In contrast, secretion of IFN-γ was similar in unfractionated versus NK1.1-depleted bone marrow CD4 + /CD8 + T cells. In comparison, sorted peripheral blood CD4 + /CD8 + T cells secreted high levels of IFN-γ and a low level of IL-4 (Table I ). Therefore, a high ratio of IFN-γ/IL-4 production correlated with the capacity to induce severe GVHD. To determine whether the high capacity of NK1.1 + bone marrow T cells to produce IL-4 was important for the negative regulation of GVHD by this cell population, bone marrow CD4 + /CD8 + T cells from wild-type and IL-4 −/− mice of the C57BL/6 background were used to induce GVHD in the irradiated BALB/c hosts. The surface phenotype and percentages of bone marrow α/β + T cells, particularly the NK1.1 + and CD4 − CD8 − T cell subsets, were similar in IL-4 −/− and wild-type mice , indicating that IL-4 is not required for NK1.1 + T cell development. The percentage of CD4 + and CD8 + NK1.1 + T cells in the IL-4 −/− mice was also similar to that in the wild type (data not shown). Whereas 2.00 × 10 5 of wild-type CD4 + /CD8 + bone marrow T cells failed to induce lethal GVHD , the addition of 1.00 × 10 5 of these bone marrow T cells from IL-4 −/− mice to the transplant induced lethal GVHD in 90% of irradiated BALB/c hosts by day 11 . Reduction of the dose of IL-4 −/− CD4 + /CD8 + T cells to 5.0 and 2.5 × 10 4 resulted in the reduction of lethal GVHD to 60 and 50%, respectively . Examination of surviving hosts at day 40 again showed histopathological evidence of GVHD of the large intestine (data not shown). Purified CD4 + and CD8 + bone marrow T cells from IL-4 −/− donors secreted no detectable IL-4, and high levels of IFN-γ that were not significantly different than that of cells from wild-type C57BL/6 mice (Table I ). Taken together, these results indicate that the NK1.1 + subset of CD4 + /CD8 + bone marrow T cells inhibited the induction of GVHD by NK1.1 − subset and that this inhibition was IL-4 dependent. We attempted to directly test the suppressive capacity of purified NK1.1 + CD4 + /CD8 + T cells on GVHD. However, as immunofluorescent staining of bone marrow T cells with anti-NK1.1 mAb, a step required for cell purification by positive selection, substantially reduced the capacity of CD4 + /CD8 + T cells to secrete IL-4 in vitro (Table I ), this approach was not pursued. As an alternative approach, we performed experiments in which purified CD4 − CD8 − α/β + T cells from the bone marrow, which are almost all NK1.1 + , were combined with NK1.1 − CD4 + /CD8 + bone marrow T cells and transplanted with TCD bone marrow. These purified bone marrow CD4 − CD8 − α/β + T cells secreted levels of IL-4 (mean 829 pg/ml) and IFN-γ (mean 1,216 pg/ml) that lacked a statistically significant difference ( P > 0.1) from that of NK1.1 + -containing bone marrow CD4 + /CD8 + T cells (Table I ). Addition of 1.00 × 10 5 CD4 − CD8 − bone marrow T cells and 2.00 × 10 5 NK1.1 − CD4 + /CD8 + bone marrow T cells to the transplant significantly increased the survival of the irradiated hosts ( P < 0.01, log rank test) as compared to the injection of only NK1.1 − CD4 + /CD8 + bone marrow T cells and TCD bone marrow . Only 10% of host mice died by day 60 in the group that received CD4 − CD8 − T cells. In contrast, the addition of 1.00 × 10 5 sorted CD4 − CD8 − bone marrow T cells from IL-4 −/− mice resulted in reduced survival, and 80% of hosts died by day 12 posttransplantation. Surviving hosts given CD4 − CD8 − bone marrow T cells from wild-type or IL-4 −/− donors were killed on day 60. Interestingly, the large intestines and skin of hosts given IL-4 −/− CD4 − CD8 − T cells showed more severe histopathological lesions of GVHD compared not only to recipients of wild-type CD4 − CD8 − T cells but also to hosts that received only NK1.1 − CD4 + /CD8 + T cells with TCD bone marrow . The sorted CD4 − CD8 − bone marrow T cells from the IL-4 −/− donors secreted a high level of IFN-γ (mean 1,024 pg/ml) that was similar to the level secreted by CD4 − CD8 − bone marrow T cells (Table I ). This indicates that the secretion of IFN-γ by NK1.1 + T cells, when it is unopposed by IL-4 secretion, exacerbates rather than inhibits GVHD. We directly compared the ability of sorted CD4 and CD8 T cells obtained from the peripheral blood or bone marrow of C57BL/6 mice to induce acute GVHD in lethally irradiated BALB/c hosts coinjected with stringently TCD C57BL/6 marrow cells. The peripheral blood T cells were at least 30-fold more potent than the marrow T cells on a per-cell basis as judged by mortality of the hosts during a 100-d observation period. In fact, no significant mortality was induced by the sorted marrow T cells in the dose range tested unless the NK1.1 + T cells were removed or the sorted marrow T cells were obtained from IL-4 −/− donors. Surprisingly, the bone marrow NK1.1 + T cells contained CD4 + , CD8 + , and CD4 − CD8 − subsets, whereas only the CD4 + and CD4 − CD8 − subsets could be detected in the thymus and spleen as reported previously ( 20 ). CD4 + and CD8 + NK1.1 + T cells in the marrow show similar cytokine secretion profiles (Zeng, D., and S. Strober, manuscript in preparation). Sorted wild-type NK1.1 + T cells (CD4 − CD8 − ) from the marrow that secreted high levels of both IFN-γ and IL-4 suppressed GVHD as judged by mortality and by histopathological changes in the skin and large intestines. In contrast, sorted IL-4 −/− NK1.1 + T cells that secreted high levels of IFN-γ without IL-4 exacerbated mortality and the histopathological changes of GVHD. The experimental results show that peripheral blood or bone marrow NK1.1 − α/β + T cells induce and NK1.1 + α/β + T cells potently suppress acute lethal GVHD and that this negative regulatory effect requires IL-4. These results agree with and extend previous studies showing that CD4 − CD8 − T cells (“natural suppressor” cells) inhibit GVHD ( 25 – 27 ), and CD4 + or CD8 + T cells that secrete a Th1-type cytokine pattern (e.g., IFN-γ and IL-2, but not IL-4) induce GVHD and those that secrete a Th2-type pattern (e.g., IL-4, but not IFN-γ and IL-2) are protective ( 34 – 36 ). However, two recent studies concluded that secretion of IFN-γ by allogeneic donor cells is not required for the induction of lethal GVHD in irradiated hosts, as donor cells from IFN-γ −/− mice induced lethal disease in either wild-type or IFN-γ −/− hosts ( 37 , 38 ). In some studies, injection of IFN-γ or IL-12 into allogeneic bone marrow transplant hosts protected against GVHD when given at the same time or shortly after the injection of the donor cells ( 39 , 40 ). The current study did not determine whether the induction of GVHD was dependent upon IFN-γ secretion by donor NK1.1 − T cells, and it is possible that alloantigen-induced secretion of other cytokines such as IL-2 is critical for the development of the disease ( 37 ). The current results suggest that NK1.1 + T cell secretion of IFN-γ in the absence of IL-4 can worsen GVHD when it is induced by donor NK1.1 − T cells, perhaps due to the specific kinetics and localization of IFN-γ secretion by the NK1.1 + T cells. GVHD induced by donor spleen cells added to TCD donor bone marrow cells has been reported to be less vigorous when IL-4 −/− rather than wild-type donor mice are used ( 37 ). The latter result appears contradictory to the increase in severity of GVHD induced by bone marrow CD4 + / CD8 + T cells from IL-4 −/− donors as compared to wild-type donors in the current study. Because the percentage of NK1.1 + T cells in the spleen is quite low and comparable to that in the peripheral blood, changes in the ability of splenic T cells from wild-type versus cytokine-deficient donors to induce GVHD are likely to reflect changes mainly in the function of NK1.1 − T cells, which recognize predominantly polymorphic MHC class I and II antigens. However, changes in the ability of bone marrow T cells from IL-4 −/− donor mice to induce GVHD are due mainly to changes in the function of NK1.1 + T cells, which recognize the nonpolymorphic CD1 antigen ( 20 ). A simple Th1/ Th2 paradigm cannot explain the capacity of all T cell subsets to induce or protect against GVHD in a variety of animal models of bone marrow transplantation, as a given cytokine such as IFN-γ can ameliorate or worsen disease depending on the preparatory regimen (lethal versus sublethal or no irradiation) used to treat the host ( 37 , 41 ). Decreased production of NK1.1 + α/β thymocytes and a resulting decrease in NK1.1 + α/β T-lineage cell–mediated IL-4 production appears to play a key role in the development of diabetes mellitus in nonobese diabetic mice, a disease which depends on NK1.1 − α/β + T cells for its development ( 22 – 24 ). Taken together with our observations, this suggests that the NK1.1 + α/β + T cell population is an important negative regulator of other α/β + T cell populations. A human CD4 − CD8 − α/β + T cell subset (V α 24-J α Q) is found in most healthy individuals and has striking similarities to the murine NK1.1 + α/β + T cell population ( 42 , 43 ). These similarities include a highly limited αβ-TCR repertoire that is restricted, at least in part, by the CD1d molecule ( 43 ). In addition, this subset expresses high levels of NKR-P1A, the human homologue of murine NK1.1 molecule, and has a high capacity to produce IL-4 and IFN-γ ( 43 ). Therefore, given our results, it will be of interest to determine if inclusion rather than depletion of donor V α 24-J α Q CD4 − CD8 − T cells in allogeneic bone marrow transplantation reduces GVHD in humans. Our results also raise the possibility that IL-4 produced by this CD4 − CD8 − T cell population or administered therapeutically might prevent or ameliorate GVHD in humans. | Study | biomedical | en | 0.999997 |
10190899 | IFN-γR knockout (KO; 27) and IFN-γR wild-type (WT) mice, both on a 129/Sv background, were bred as homozygotes from breeders initially provided by Maria Wysocka (Wistar Institute, Philadelphia, PA). TNFR p55/p75–deficient mice ( 28 , 29 ) on a mixed 129/Sv × C57BL/6 background were bred in our facility using homozygous breeding pairs provided by Mark Moore ( Genentech, Inc. , South San Francisco, CA). iNOS-deficient mice ( 30 ) on a second generation C57BL/6 backcross were bred through a National Institutes of Allergy and Infectious Diseases contract with Taconic Farms, Inc. C57BL/6 and C57BL/6 × 129/SvEv F1 hybrids obtained from the The Jackson Laboratory were used as WT controls for the iNOS- and TNFR-deficient mice, respectively. Donor (at least 8 wk old) and recipient animals were sex matched. Recipient mice were given lethal total body irradiation (950–1,000 rads) and reconstituted intravenously with 10–20 million bone marrow cells within 24 h. Marrow cell suspensions were prepared from donor tibial and femoral bones by flushing with RPMI 1640 ( GIBCO BRL ) supplemented with antibiotics (penicillin [100 U/ml] and streptomycin [100 U/ml]) using a 25-gauge needle syringe. Irradiated and reconstituted mice were given Bactrim (sulfamethoxazole [150 mg/ml] and N -trimethoprim [30 mg/ml]; Teva Pharmaceuticals) in their drinking water for 5 wk. Thereafter, they were switched to sterile drinking water, thus ensuring that the antibiotic treatment would not affect the ensuing experimental infection with T . gondii . Unless otherwise stated, mice were used for experimental infection or for analysis of chimerism 8–9 wk after BM cell transfer. For each infection experiment, groups of nonirradiated WT and KO animals were included as positive and negative controls for host resistance, respectively. In every instance except one, sham chimeric KO→ KO mice exhibited mortality rates identical to those of nonirradiated KO mice, whereas WT→ WT mice behaved like nonirradiated WT mice. However, sham chimeric mice that were used as controls for the studies involving iNOS- deficient mice reproducibly (in three independent experiments) succumbed ∼30 d after infection, much earlier than unmanipulated C57BL/6 mice, which survived for at least 60 d. The reason for the earlier mortality of the C57BL/6 sham chimeras is unclear. These mice did not succumb unless infected with T . gondii and showed full reconstitution of splenic lymphoid and myeloid cell populations as determined by flow cytometric analysis (data not shown). The extent of hemopoietic cell replacement by donor phenotype cells upon reconstitution was analyzed 8 wk after transfer of BM cells using mice chimeric for iNOS gene deficiency. Spleen cells and d5 thioglycollate–elicited peritoneal cells were harvested from each of three mice per group. Cells were plated in 96-well plates at a concentration of 2 × 10 6 cells/ml and stimulated with 100 U/ml of IFN-γ and 10 μg/ml of soluble tachyzoite antigen (STAg). The culture medium consisted of RPMI 1640 ( GIBCO BRL ) supplemented with 10% FBS (Hyclone), penicillin (100 U/ml) and streptomycin (100 U/ml), glutamine ( GIBCO BRL ), and 2-ME (5 μM; Sigma Chemical Co. ). Nitrite production after 24 h was evaluated by the Griess reaction as previously detailed ( 24 ). IFN-γR KO and WT mice were immunized with 10 6 lethally irradiated (15 Krads) tachyzoites of T . gondii (RH strain). 14 d later, IFN-γ and IL-12 p40 production by spleen cells from vaccinated and naive mice was assessed. Spleen cell cultures (3 × 10 6 /ml) were stimulated with either ConA (5 μg/ml) or STAg (10 μg/ml). Supernatants were harvested 24 h later for IL-12 p40 determination and 48 h later for IFN-γ measurement. Previously described ELISA protocols were used to measure IL-12 p40 and IFN-γ levels in culture supernatants ( 22 ). Tachyzoites of the RH strain of T . gondii were cultivated in human foreskin fibroblasts maintained in DMEM ( GIBCO BRL ) supplemented with 1% FCS and antibiotics. The ME49 strain of T . gondii was passaged as cysts in C57BL/6 mice. Experimental animals were infected with 20 ME49 cysts by intraperitoneal injection. An additional set of IFN-γR chimeric mice were infected intraperitoneally with 33 L . monocytogenes (EGD strain) bacteria, a dose equivalent to 1/100 of LD 50 for IFNR WT mice. The L . monocytogenes inoculum was prepared from a frozen stock. Bacterial counts were confirmed by plating serial dilutions of the stock on Mueller–Hinton agar plates on the day of infection. Measurement of the extent of replacement of tissue macrophages by donor-derived cells is required for proper interpretation of the results obtained with BM chimeric mice. We performed this assessment in iNOS BM chimeric mice, in which IFN-γ– and STAg-induced NO production can be used as a readout of macrophage function. 8 wk after reconstitution, the ability of spleen cells to produce NO in response to IFN-γ and parasite antigen was clearly dictated by the genotype of the BM donor . For instance, spleen cells from iNOS KO mice reconstituted with WT marrow exhibited robust NO production. Importantly, there was no residual NO-producing capacity detectable in WT mice reconstituted with iNOS KO BM. Similarly, the capacity of thioglycollate-elicited peritoneal cells to produce NO in response to IFN-γ and STAg stimulation was dictated by the BM donor genotype . Identical results were obtained using chimeric IFN-γR KO mice (data not shown). Thus, in these chimeras, most if not all of the macrophages in a lymphoid organ (spleen) or at a site of cellular recruitment (peritoneal cavity) appear to consist of donor-derived cells. Having demonstrated successful and complete reconstitution of macrophages by donor BM-derived cells, we proceeded to construct chimeric mice using WT and IFN-γR KO on the same 129/SvEv genetic background. IFN-γR KO mice fail to respond to IFN-γ and exhibit increased susceptibility to a variety of intracellular pathogens ( 27 , 31 ). Both the cis and trans models predict that a KO→ WT chimera should exhibit susceptibility to infection because the BM-derived elements, including macrophages and neutrophils, will not be able to control infection. More importantly, however, different outcomes are predicted by the two models for the WT BM→ KO chimera. The trans model predicts resistance for this chimera, because the WT marrow-derived cells would be armed and protect somatic cells, whereas the cis model predicts susceptibility for this chimera, because the somatic cells would be unable to control infection due to a lack of IFN-γ responsiveness. An assumption made for the KO→ WT chimera is that the KO BM-derived cells remain competent for IFN-γ production in the absence of responsiveness to IFN-γ. Taking into account previous reports of IFN-γ amplification of the IL-12 response ( 32 , 33 ), a concern raised by these observations is that type 1 responses may not develop normally in the absence of IFN-γ responsiveness in APC and T cell populations. To address these concerns, spleen cells from uninfected IFN-γR KO and WT mice were stimulated with tachyzoite extract (STAg), and their capacity to produce IL-12 p40 as well as IFN-γ was measured by ELISA. As shown in Fig. 3 , A and B, production of both cytokines in response to STAg stimulation was not compromised by IFN-γR deficiency. To assess possible defects in type 1 cell development, IFN-γR KO mice were immunized with irradiated tachyzoites and their spleen cells restimulated with STAg or mitogen in vitro. Robust, antigen-specific IFN-γ production was observed in cultures from both WT and KO mice . On the basis of these control experiments, we predicted that in WT mice reconstituted with IFN-γ–unresponsive BM cells, IL-12–dependent NK as well as T cell IFN-γ production should not be impaired. As expected, sham (KO→ KO) chimeric mice completely deficient in IFN-γR were acutely susceptible to T . gondii infection, whereas WT→ WT sham chimeric mice survived infection for at least 60 d . As predicted by both trans and cis models, chimeric WT mice reconstituted with IFN-γR–deficient BM were susceptible to acute T . gondii infection. Importantly, however, in WT→ KO chimeras, IFN-γR deficiency in the recipient compartment, despite a WT BM donor genotype, resulted in acute mortality. This observation indicates that IFN-γ activation of WT BM-derived cells is not sufficient to confer protection and that nonhemopoietic cells responsive to this lymphokine are required, an interpretation consistent with the cis model. A possible caveat is that at week eight, WT→ KO BM reconstitution may be incomplete in tissue sites other than the spleen and the peritoneum, where chimerism was initially assessed . The latter explanation is unlikely, however, because even after an additional 2 mo of reconstitution, these animals remained fully susceptible to acute infection (data not shown). To further confirm that full functional reconstitution was achieved after 8 wk, similarly constructed sets of chimeric mice were infected with the macrophage-trophic intracellular bacterium L. monocytogenes and survival of chimeric mice assessed after challenge with a sublethal dose. In the case of this pathogen, IFN-γR expression on the BM but not in the somatic cell compartment was required for resistance . The latter finding confirms that after only 2 mo, WT BM reconstitution of IFN-γR KO recipients is sufficiently complete to confer potential resistance to intracellular infections. The above experiments, by demonstrating a differential requirement for IFN-γR expression on somatic cells for protection against Toxoplasma versus Listeria , underscore the critical importance of host cellular niches in determining the effector cell types and mechanisms required for control of these pathogens. Resistance to T . gondii is exquisitely dependent on IFN-γ, not only during acute infection but also during the chronic phase of infection ( 20 , 21 ). For instance, administration of neutralizing antibodies to IFN-γ 30 d after inoculation allows uncontrolled cyst reactivation and tachyzoite replication in the brain parenchyma and invariably causes death of the host within 10 d. To further explore the role of nonhemopoietic cells versus BM-derived inflammatory cells as effectors in the chronic phase of the infection using the IFN-γR KO chimeric mice, partial chemotherapy (commencing 3 d after parasite inoculation) was employed to allow host survival through the acute phase and establishment of persistent infection (as evidenced by cyst formation in brain tissue). Once infection was established (at day 20), further drug treatment was terminated and the survival of the chimeric mice monitored. As shown in Fig. 5 , even with oral drug treatment, a majority of sham IFN-γR KO chimeric mice succumbed to infection by week three postinfection. Chimeric mice with IFN-γR deficiency in either the hemopoietic or nonhemopoietic compartments also died within 5–11 d following cessation of chemotherapy. The kinetics of mortality in these two sets of chimeric mice were essentially identical. As expected, WT sham chimeric mice survived the infection even after drug treatment was withheld. Thus, as observed for resistance to acute infection, IFN-γR expression on nonhemopoietic as well as BM-derived cells is essential for host survival in established infection, as assessed in this drug treatment model. Although IFN-γ–mediated signals are clearly important and required on both somatic and hemopoietic cells during the acute and chronic phases of infection, IFN-γ alone does not suffice, at least during the chronic phase. At this stage, TNF-α is also crucially required for host resistance. Thus, administration of anti-TNF Ab (without additional neutralization of IFN-γ) is sufficient to reactivate infection and induce lethal encephalitis in chronically exposed C57BL/6 mice ( 21 ). Additionally, T . gondii infections are lethal in mice lacking either the TNF p55 receptor or both the p55 and p75 receptors ( 23 , 24 ). In the case of this as opposed to IFN-γR deficiency, lethality occurs only after infection establishes in the CNS (∼3 wk after infection). Because TNFRs are expressed not only on macrophages and other hemopoietic cells but also on nonhemopoietic cells, including astrocytes and neuronal cells, we evaluated the requirement for TNFR signaling on these cellular compartments for resistance to chronic infection. Reciprocal chimeras were constructed between WT (B6 × 129/J F1) and TNFR p55/p75 KO mice on the same hybrid background. As shown in Fig. 6 , TNFR KO mice reconstituted with KO BM cells succumbed to infection within 20 d, as previously reported for unmanipulated TNFR KO mice. WT controls and WT→ WT sham chimeric mice survived T . gondii infection for at least 60 d. However, TNFR KO BM cells in the context of a WT recipient did not confer the same susceptibility phenotype observed in completely deficient mice. Similarly, WT BM→ KO chimeras also exhibited only partial resistance to chronic infection. Thus, both compartments must be receptor deficient or WT to exhibit a completely susceptible or resistant phenotype, respectively. In contrast to these findings, resistance to Listeria monocytogenes has been shown to require TNFR expression only on BM-derived cells ( 34 ). These divergent requirements for TNFR expression in Toxoplasma and Listeria systems further highlight the importance of the parasitized cellular niche in determining which effector cell populations are required for host resistance. We have previously proposed that TNFRs expressed on nonhemopoietic, somatic cells such as neurons and astrocytes may be important in activating them to control intracellular tachyzoite replication by an iNOS-independent mechanism ( 24 ). This hypothesis was based on the finding that although TNFR (p55/p75) KO mice develop necrotizing encephalitis and die at the same rate as iNOS-deficient mice, they nevertheless display significant iNOS induction in brain tissue. Because macrophages from the same TNFR KO mice can exhibit both NO production and microbicidal activity in vitro and ex vivo, these observations suggested that the defect responsible for death of the infected TNFR-deficient animals resides in nonhemopoietic effector cells. The intermediate level of resistance displayed by KO→ WT and WT→ KO TNFR chimeric mice argues instead that control of chronic infection may require TNFR expression on both hemopoietic and nonhemopoietic compartments. If the extreme susceptibility of the TNFR KO mice is explainable solely by iNOS deficiency, then the resistance patterns in iNOS chimeric mice should be identical to those exhibited by TNFR chimeric animals. As shown in Fig. 7 , this is clearly not the case. In contrast to the parallel TNFR-deficient chimera, iNOS→ WT BM chimeric mice displayed susceptibility indistinguishable from either iNOS→ iNOS or nonirradiated iNOS KO mice . Furthermore, the WT→ KO reciprocal chimera exhibited a survival pattern virtually indistinguishable from the control WT→ WT chimera. For reasons that are not clear (see Materials and Methods), these sham chimeric mice succumbed early, at ∼30 d, in contrast to nonirradiated WT C57BL/6 mice, which, as previously reported ( 26 ), survived greater than 60 d . Nevertheless, the findings clearly indicate that, in the iNOS system, the BM genotype is the main determinant of the resistance phenotype of the chimera. This is in direct contrast to the situation in both IFN-γR and TNFR chimeric animals, in which both hemopoietic and nonhemopoietic cells contribute to host resistance during the chronic phase of infection. Taken together, these results indicate that the trans mechanism, whereby IFN-γ– and TNF-α–dependent production of toxic metabolites such as NO by mononuclear phagocytes is, by itself, not sufficient to account for immune control of a pathogen that infects both hemopoietic and nonhemopoietic cells. Instead, our findings are more consistent with the cis model of host resistance against intracellular pathogens, which proposes that nonhemopoietic cells need to be directly activated by lymphokines and play an active role as effectors of IFN-γ/TNF-α–dependent CMI to T . gondii . The latter requirement may explain why, although not essential for cell survival or tissue homeostasis ( 24 , 27 – 29 ), the expression of IFN-γR and TNFR has been retained in all nucleated cell types during the course of evolution ( 35 , 36 ). Nevertheless, it remains unclear whether or not the direct activation of nonhemopoietic cells by IFN-γ and TNF-α is sufficient to completely restrict parasite growth within these cells. Although in vitro experiments have clearly documented that nonhemopoietic cells can do so in the absence of macrophages ( 11 ), our in vivo results do not exclude potential synergism between trans-acting diffusible metabolites such as NO derived from hemopoietic cells and the cis-acting IFN-γ/TNF-α–dependent mechanism in limiting tachyzoite replication within nonhemopoietic cells. The identical susceptibility observed in iNOS→ iNOS and iNOS→ WT chimeric mice underscores the critical importance of NO production by cells derived from the hemopoietic lineage in host resistance. This conclusion agrees with previous reports of the iNOS dependence of the antitoxoplasmic activity of lymphokine-activated macrophages and microglial cells ( 37 , 38 ). In parallel, the comparable survival curves exhibited by either iNOS-deficient or WT recipients reconstituted with WT BM suggests that iNOS expression in the nonhemopoietic cell compartment plays a limited if not minimal role relative to that in hemopoietic cells. Consistent with this conclusion is the recent observation that lymphokine-induced control of intracellular tachyzoite growth occurs effectively in astrocytes (putative nonhemopoietic effector cells in the brain) derived from iNOS-deficient mice ( 39 ). Nevertheless, because of technical problems uniquely observed in the iNOS BM chimera experiments, we cannot definitively conclude that iNOS expression in the nonhemopoietic compartment is absolutely without antimicrobial function. Thus, the premature mortality exhibited by sham chimeric WT mice suggests that lethal irradiation may have damaged a subset of nonhemopoietic cells required for long-term survival after infection and could have, in theory, masked any protective effect of iNOS in the nonhemopoietic compartment. Notwithstanding, it is reasonable to conclude, based on the marked differences in mortality observed between iNOS→ WT and WT→ iNOS animals, that the IFN-γ– and TNF-α– dependent resistance mechanism(s) operating within nonhemopoietic cells has a major iNOS-independent component. The nature of the cis-acting effector mechanism(s) responsible for restricting the growth of T . gondii within nonhemopoietic cells is presently undefined. A primary candidate mechanism is the depletion of intracellular tryptophan stores by the enzyme indoleamine dioxygenase (IDO; 12). IFN-γ and TNF-α synergistically induce the transcription and activation of this enzyme in many human cell types, including fibroblasts, retinal pigmented epithelium, and neurons as well as macrophages ( 12 , 40 – 42 ). Nonetheless, the evidence for the importance and contribution of the IDO mechanism to host resistance in murine cells is more tenuous and controversial than in human cells ( 43 ). IFN-γ reportedly fails to induce IDO and toxoplasmastatic activity in mouse fibroblasts ( 44 ). Furthermore, in murine macrophages, NO induction by IFN-γ results in cross-inhibition of IDO gene transcription and enzymatic activity ( 42 , 45 ). The above observations suggest that other as yet unidentified iNOS- and IDO-independent mechanism(s) are responsible for resistance to T . gondii infection, a conclusion also reached in a study involving IFN-γ–induced control of the parasite by endothelial cells ( 46 ). The existence of iNOS-independent, IFN-γ-dependent mechanisms of host resistance is not unique to T . gondii infection. A similar divergence in the resistance phenotypes of IFN-γ– and iNOS-deficient mice has been described in Chlamydia infections ( 47 ). In the case of these two pathogens, in vitro transfection experiments have directly implicated the IDO pathway in the control of microbial growth within nonhemopoietic cells ( 48 , 49 ). An IFN-γ/TNF-α– dependent but iNOS-independent mechanism of CD8 T cell–mediated host resistance has also been described for hepatitis B infection, based on adoptive transfer experiments in a transgenic mouse model ( 50 ). Clearly, the identification of these important and potentially novel effector pathways is a highly relevant area for future investigation and represents a major frontier for the field of microbial immunity. | Study | biomedical | en | 0.999997 |
10190900 | The NK cell lines NK3.3 (a gift from J. Kornbluth, St. Louis University School of Medicine, St. Louis, MO) and NK-92 (obtained from H.-G. Klingemann, Rush University, Chicago, IL) were cultured as previously described ( 16 , 17 ). HLA class I transfectants of the 721.221 cell line were obtained from J. Gumperz and P. Parham (Stanford University, Stanford, CA), except for the HLA-G transfectant, which was obtained from L. Lanier (DNAX, Palo Alto, CA). The T leukemic cell line Jurkat and the monocytic cell line HL60 were obtained from the American Type Culture Collection. RMA-S cells transfected with HLA-E (RMA-S-E) cells were obtained from E.H. Weiss and M. Ulbrecht (Institut für Anthropologie and Humangenetik der Universität München, Munich, Germany) and used as previously described ( 3 ). NK clones were produced from the peripheral blood of normal donors as previously described ( 18 ). Anti-CD3 and anti-CD56 antibodies conjugated to PE were purchased from Becton Dickinson . The HLA-specific mAb B9.12.1, the KIR2DL1-reactive mAb EB6, and isotype-matched control Abs were obtained from Immunotech. Rabbit antisera to the NH 2 -terminal region of KIR2DL4 (referred to as anti-2DL4) were raised by immunization with the synthetic peptide VGGQDLPFC, and were affinity purified on the same peptide (Research Genetics). The mAb VV1-IG10, specific for the A33 early/intermediate vaccinia protein, was a gift of A. Schmaljohn (U.S. Army, Fort Detrick, Frederick, MD). The MHC class I–specific mAb DX17 was a gift of L. Lanier. The HLA-G–specific mAb (G233) was a gift of A. King (Cambridge University, Cambridge, UK). This mAb does not react with classical HLA molecules and reacts with HLA-G in extravillous trophoblasts ( 19 ). The KIR2DL4– Ig fusion protein was produced by the same strategy used for other KIR–Ig proteins ( 20 ). The extracellular region was PCR amplified from a cDNA clone of KIR2DL4 obtained from A. Selvakumar and B. Dupont (Memorial Sloan Kettering Cancer Center, New York, NY) with the forward primer CAGAGTGTGCTAGCGCACGTGGGTGGTCAGGACAAGCC containing an NheI site, and the reverse primer GAGTACCTAGGATCCGCATGCAGGTGTCTGGCGATACC containing a BamHI site. These PCR fragments were cloned into the expression vector Cd5lneg1 (obtained from B. Seed, Massachusetts General Hospital, Charlestown, MA). SDS-PAGE analysis of the purified recombinant protein identified a species of ∼65 kD under reducing conditions. The binding assay was performed as previously described ( 20 ), except that the cells were incubated with goat IgG (50 μg/ml) for 30 min after incubation with the 2DL4–Ig fusion protein and before addition of the FITC-conjugated goat anti–human Fc. Binding was assessed by flow cytometry. cDNAs encoding KIR2DL4 and NKG2A (obtained from J. Houchins, R&D Systems, Minneapolis, MN) were subcloned into the plasmid pSC65 and used to generate recombinant vaccinia viruses as previously described ( 21 ). Purified viruses encoding KIR2DL4 or NKG2A were used to infect the human cell line NK-92, as previously described ( 20 ). Vaccinia virus infections were monitored by flow cytometry with the mAb VV1-IG10. Infected and uninfected control cells were simultaneously plated for standard 51 Cr-release assays and for Ab staining followed by flow cytometry as previously described ( 20 ). Peptide loading was done as previously described ( 3 ). In brief, 500 μM of the HLA-G signal sequence peptide (VMAPRTFL) was incubated overnight with RMA-S-E cells plated at 5 × 10 5 cells/ml. Cells were washed and used for antibody staining followed by flow cytometry. A soluble recombinant protein containing the extracellular portion of KIR2DL4 fused to the Fc region of human IgG1 (KIR2DL4–Ig) was produced in order to search for its ligand. Binding of KIR2DL4–Ig to a panel of HLA-transfected 721.221 cells was analyzed by flow cytometry. KIR2DL4–Ig displayed a uniform binding to all the 721.221 transfectants tested, as well as untransfected 721.221 cells . This HLA class I–independent binding of KIR2DL4 to 721.221 cells may be due to the first Ig domain (D0), as similar results have been reported with soluble KIR3DL1 ( 22 ) and KIR3DL2 ( 23 ), both of which contain a D0 domain, and with a soluble D0–Ig fusion protein ( 22 ). In contrast, KIR2DL4–Ig bound strongly to 721.221 cells expressing HLA-G (221–G). As expected, KIR2DL2–Ig bound to its ligand HLA-Cw3 but not to HLA-G expressed on 721.221 cells . Unlike previous studies describing weak and heterogeneous binding of the similar p49 KIR ( 15 ) and the Ig-like transcript (ILT)-2 and ILT-4 members of the ILT inhibitory receptor family expressed mainly on monocytic cells ( 24 , 25 ), binding of KIR2DL4–Ig to HLA-G was detected by flow cytometry as a bright and uniform peak . The panel of HLA transfectants included HLA-A1, -A2, -B7, -Cw3 and -G, all permissive for HLA-E expression, and HLA-B46, -B51, and -B58, which are not permissive for HLA-E expression . Thus, it is unlikely that the KIR2DL4–Ig binds to the HLA-E molecules that reach the cell surface upon binding the peptide derived from the HLA-G leader sequence. The comparison of KIR2DL4–Ig binding to 221–B7 and 221–G cells provides further evidence that KIR2DL4 is not binding to HLA-E. The peptide from the HLA-B7 leader sequence binds HLA-E fivefold better than the HLA-G–derived leader peptide in an in vitro peptide binding assay, resulting in higher HLA-E surface expression ( 2 ). Yet despite a very high surface level of HLA-B7 expression on 221–B7 cells, there was no binding of KIR2DL4–Ig to 221–B7 above the HLA-independent binding . The KIR2DL4 gene is transcribed in every NK cell tested ( 15 , 26 ), but there is no information on protein expression of KIR2DL4 in NK cells. An antiserum against a peptide corresponding to a unique NH 2 -terminal sequence of KIR2DL4 was produced to examine cell surface expression of KIR2DL4 on a panel of NK, T, B, and monocyte/macrophage cell lines. The anti-KIR2DL4 (anti-2DL4) antiserum reacted with the NK cell lines NK-92 and NK3.3. . There was negligible staining of cell lines such as the T leukemic line Jurkat, the B cell line 721.221, and the monocytic cell line HL-60. All NK clones in a random panel tested ( n = 14) expressed uniformly high cell surface KIR2DL4 . To test whether KIR2DL4 was expressed on all NK cells, CD3 − CD56 + cells were isolated from the peripheral blood lymphocytes of four donors. All CD56 + cells (>99%) in these cultures reacted with the anti-2DL4 antiserum . In contrast, only 2% of the CD3 + lymphocytes reacted with the anti-2DL4 antiserum . The proportion of KIR2DL4 + cells within the CD3 + population varied between 1 and 9% among six donors tested. Three-color analysis of the CD3 + 2DL4 + subpopulation by flow cytometry showed that the majority of these cells also expressed the NK marker CD56 (data not shown). A recombinant vaccinia virus encoding KIR2DL4 (vac-2DL4) was produced for functional transfer experiments in order to confirm the ligand specificity seen in binding studies and to test whether KIR2DL4 can inhibit the lysis of HLA-G– bearing targets. The highly lytic NK tumor cell line NK-92, chosen because it has been used successfully for vaccinia virus-mediated expression ( 18 , 20 ), was tested for the inhibition of lysis of 721.221 target cells expressing different HLA class I molecules. NK-92 cells express a low endogenous level of KIR2DL4 as determined by flow cytometry . In multiple experiments ( n = 30), lysis of the HLA-G–expressing target cell 221–G was reduced to 73 ± 19% of the lysis observed with the HLA class I–deficient parental cell 721.221. This low but reproducible inhibition would be consistent with recognition of HLA-G by endogenous KIR2DL4. The level of expression of KIR2DL4 on infected NK-92 was very similar to that of endogenous KIR2DL4 on NK clones . Vaccinia virus–infected NK-92 cells expressing KIR2DL4 lysed 721.221 cells and 721.221 cells transfected with HLA-Cw4 . In contrast, there was striking inhibition of lysis of 221–G cells. A number of other 721.221 transfectants expressing HLA class I genes, such as HLA-A1, -A2, -B7, -B58, -Cw3, and -Cw7, were not protected from lysis by NK-92 cells expressing KIR2DL4 (Table I , and data not shown). If 221–G cells are protected from lysis through recognition of HLA-G by KIR2DL4, lysis should be restored in the presence of anti–HLA class I antibodies such as DX17 ( 27 ). KIR2DL4-expressing NK-92 cells incubated with target cells in the presence of DX17 mAb lysed the 221–G cells to the same extent as untransfected 721.221 cells . Several HLA class I molecules, including HLA-G, are permissive for the expression of HLA-E ( 2 – 4 ). However, lysis of the HLA-E–permissive 221–Cw4 cells by NK cells expressing KIR2DL4 suggests that HLA-E is not recognized by KIR2DL4. To further distinguish between HLA-G– and HLA-E–mediated protection, NK-92 cells expressing either KIR2DL4 or the HLA-E–specific inhibitory receptor CD94/NKG2A were tested against a panel of 721.221 cells transfected with individual HLA class I genes. The endogenous CD94/NKG2A present on NK-92 cells is not sufficient to provide inhibition of lysis upon recognition of HLA-E–expressing target cells (Table I ). However, increasing the cell surface expression of NKG2A using a recombinant vaccinia virus (as detected by flow cytometry; data not shown) resulted in inhibition of lysis that correlated with HLA-E expression (Table I ). Thus, there was inhibition of lysis of target cells expressing HLA-A1, -Cw3, -Cw4, and -G, but not HLA-B58, an allele not permissive for HLA-E expression (Table I ). It is worth noting that the complete inhibition of 221–G target cell lysis by NK-92 infected with vac-NKG2A may reflect the combined inhibition mediated by recognition of HLA-E by CD94/NKG2A and of HLA-G by endogenous KIR2DL4. In contrast, as reported above, NK-92 cells expressing KIR2DL4 were inhibited only by HLA-G–expressing cells and not by cells expressing other HLA class I molecules along with HLA-E. These data show that functional transfer of KIR2DL4 into NK-92 cells conferred specificity for HLA-G, leading to inhibition of target cell lysis. Finally, to obtain independent evidence that HLA-G and not HLA-E is recognized by KIR2DL4, we tested the effect of an HLA-G–specific mAb on the KIR2DL4-mediated inhibition of NK-92. To test whether this mAb may recognize HLA-E in the context of a bound peptide derived from the HLA-G signal peptide, the transporter for antigen presentation (TAP)-deficient mouse RMA-S cell transfected with HLA-E (denoted RMAS-E) was loaded with the HLA-G signal sequence peptide VMAPRTLFL at 26°C. This resulted in increased surface stabilization of HLA-E as detected with the anti-HLA mAb B9.12.1. However, no binding of mAb G233 was detected . Thus, mAb G233 does not recognize HLA-E. NK-92 cells were infected with recombinant vaccinia viruses encoding KIR2DL1 or KIR2DL4, resulting in high surface expression of these receptors . Lysis of 221–G target cells was tested in the presence of G233 mAb or an isotype-matched IgG2a. Only G233 restored the lysis of 221–G cells by KIR2DL4-expressing NK-92 cells to the level observed with KIR2DL1-expressing NK-92 . mAb G233 had no effect on the inhibition of lysis of 221–Cw4 cells by KIR2DL1- expressing NK-92 cells (data not shown). These data demonstrate that the single ITIM in the context of the KIR2DL4 molecule can deliver an inhibitory signal in NK-92 cells. Experiments to test the possibility that KIR2DL4 may interact with other proteins via the positively charged arginine residue in the transmembrane domain have been hampered by the lack of an anti-KIR2DL4 antibody suitable for immunoprecipitations. It is possible that the inhibitory activity of KIR2DL4, clearly evident in the cell line NK-92, could be modulated in other cells by association with ITAM-bearing molecules such as DAP12 (which associates with KIR2DS, CD94/ NKG2C, and Ly49D/H; references 12 , 28 , 29 ) or FcRγ (which associates with ILT-1 and NKR-P1; references 30 , 31 ). In this regard, it is interesting that the cell line NK3.3 and some NK clones are not inhibited by HLA-G despite expression of KIR2DL4 (6, 8, 32, and our unpublished observations). In conclusion, these results clearly identify KIR2DL4 as a specific receptor for HLA-G, by both direct binding and functional transfer. Previously, the only NK receptor that reproducibly inhibited the lysis of HLA-G–expressing cells by NK cells was the CD94/NKG2A heterodimer ( 6 – 8 ). However, this inhibition can be explained by the binding of CD94/NKG2A to the class Ib molecule HLA-E. Moreover, CD94/NKG2A is not expressed by all NK cells ( 26 , 33 ), and CD94 − NK cells that are inhibited by HLA-G have been reported ( 6 – 8 ), suggesting the existence of yet another NK receptor specific for HLA-G. The ILT2 receptor expressed on monocytes and on a subset of NK cells can also inhibit lysis of target cells expressing HLA-G ( 34 ). However, in contrast to KIR2DL4, ILT2 and CD94/ NKG2A are not expressed by all NK cells. The basis for the immune privilege of the fetus, which is a hemi-allogeneic graft, represents an interesting immunological puzzle. Trophoblast cells do not express HLA-A or HLA-B molecules on their cell surface, a feature thought to confer protection from T cell responses. In contrast, expression of HLA-G on trophoblast cells may result in a functional interaction with KIR2DL4 on maternal decidual NK cells. CD16 − CD56 bright NK cells constitute the major population of lymphocytes in the decidua ( 13 ). The outcome of this interaction in situ remains to be established and might involve the regulation of a number of NK functions such as cytotoxicity, cytokine production, or proliferation. For example, the high expression of HLA-G on cytotrophoblasts may play a role in preventing local activation of maternal NK cells. This may provide a basis for earlier observations showing that cultured fetal trophoblast cells are resistant to lysis by NK cells isolated from either human decidua or peripheral blood ( 35 ). Alternatively, recognition of HLA-G by KIR2DL4-expressing NK cells might regulate trophoblast differentiation or invasion into the maternal decidua. Delineating the biological significance of the HLA-G–KIR2DL4 interaction at the maternal–fetal interface will be a step towards resolving the apparent immunological paradox of a successful pregnancy. | Study | biomedical | en | 0.999997 |
10190901 | HCC sera were obtained from 95 subjects included in an epidemiological study previously described and were from Henan Province, People's Republic of China ( 31 ). Sera from 77 patients with liver diseases (26 asymptomatic HBsAg carriers, 31 patients with acute hepatitis, and 20 patients with chronic hepatitis and liver cirrhosis), and 30 normal human sera, all from the same province, were available for these studies. All of the above sera came from the Sanitary and Anti-Epidemic Station (Henan Province). 40 normal human sera from the San Diego, CA area were also included as controls. Human prototype sera containing autoantibodies to previously identified intracellular antigens were from patients with systemic autoimmune diseases ( 2 ) and obtained from the serum bank of the Autoimmune Diseases Center of The Scripps Research Institute (La Jolla, CA). MOLT-4 (human acute lymphoblastic leukemia), T24 (human transitional cell bladder carcinoma), HEp-2 (human epidermoid laryngeal carcinoma), HeLa (human epitheloid cervical carcinoma), HepG2 (human hepatocellular carcinoma), A549 (human lung carcinoma), and 3T3 (mouse fibroblast) cell lines were obtained from the American Type Culture Collection and cultured following the specific protocol for each cell line. Cells grown in monolayers were solubilized directly in Laemmli's sample buffer containing protease inhibitors ( Boehringer Mannheim ). Solubilized lysates were briefly sonicated before electrophoresis on SDS–polyacrylamide gels. Initial identification of autoantibodies in sera was performed using methanol- and acetone-fixed commercial HEp-2 cell slides (Bion Enterprises, Ltd.). The findings were usually confirmed in other experiments using T24, HepG2, and 3T3 cells that were grown on coverslips, fixed for 5 min at −20°C in 100% methanol, and permeabilized for 3 min at −20°C in 100% acetone. As a second antibody, FITC-conjugated goat anti–human IgG (Caltag Laboratories) was applied. A titer of >1:40 dilution was interpreted as positive. Western blotting was performed essentially as described by Chan and Pollard ( 32 ). Cell extracts were electrophoresed on SDS-PAGE and transferred to nitrocellulose paper. After preblocking with PBS containing 0.5% Tween-20 and 5% nonfat milk for 30 min at room temperature, the nitrocellulose strips were incubated for 60 min at room temperature with a 1:100 dilution of serum. As secondary antibody, horseradish peroxidase–conjugated goat anti–human IgG (Caltag Laboratories) was applied (1:2,000 dilution). The detection of immunoreactive bands was performed with an ECL kit ( Amersham Corp. ) according to the manufacturer's instructions and followed by autoradiography. T24 and HeLa cells were cultured and radiolabeled with [ 35 S]methionine. For preparation of T24 and HeLa cell extracts, cells were collected by centrifugation, combined with two times packed cell volume buffer A (10 mM Tris-HCl, pH 7.5; 150 mM NaCl, 1.5 mM MgCl 2 , and 0.5% NP-40), and held on ice for 10 min to allow cell lysis. The supernatant obtained by centrifugation at 10,000 g for 10 min at 4°C was used as antigen preparation in immunoprecipitation studies. Before immunoprecipitation, labeled cell extracts were precleared by adding 100 μl 10% protein A–Sepharose stock/ml extract, mixed for 5 min on ice, and centrifuged to collect supernatant. Typically, 100 μl 10% protein A–Sepharose, 500 μl buffer B (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 0.5% NP-40; 0.5% deoxycholic acid; 0.1% SDS; and 0.02% sodium azide) containing BSA at 10 μl (stock: 10 mg/ml), 40 μl labeled cell extract, 10 μl serum, and 10 μl protease inhibitor ( Boehringer Mannheim ) was added to a standard immunoprecipitation reaction. After incubation for 1 h, the immunoprecipitated beads were washed five times with 1 ml buffer B. Finally, the beads were eluted with an equal volume of 2× Laemmli's sample buffer and analyzed in SDS-PAGE followed by autoradiography. HCC serum YZ was diluted 1:100 and used for screening a T24 λzap cDNA expression library. Before screening of the cDNA library, the serum was extensively absorbed against bacteria infected with wild-type λzap phage ( 33 ). The preabsorbed serum was used to immunoscreen 3.0 × 10 5 recombinant plaques using 125 I-labeled goat anti–human IgG as the secondary detecting reagent. Screening was carried out on duplicate filters, and one double-positive clone, JY1, was isolated and subcloned in vivo into pBK-CMV plasmid using ExAssist helper phage (Stratagene Inc.) as recommended in the manufacturer's instructions. The clone JY1 was amplified, purified, and used for sequence analysis. cDNA insert was analyzed by restriction mapping and sequencing. The clone JY1 was a partial sequence, and RACE methodology was used to obtain overlapping 5′ clones using the Marathon-Ready cDNA from human colorectal adenocarcinoma SW480 cell line ( Clontech ). Nucleotide sequence was determined in both strands using a semiautomated sequencer from Applied Biosystems (model 373). Oligonucleotide primers were synthesized with a DNA synthesizer (Applied Biosystems; model 394). DNA and protein sequences were analyzed by the Genetics Computer Group Sequence Analysis Software Package for UNIX computers (version 7.4; 34). Alignment of protein sequences was achieved with a Multiple Alignment Program . The ORF of p62 was reamplified and confirmed by RT-PCR using T24 cell mRNA as template. One set of sense and antisense primers was designed, and their positions with respect to the p62 full length cDNA are indicated : rt3 sense, 5′-TT GAATTC GCCATGGTGAACAAGCTTTACATCGGGAACC-3′ and rt4 antisense, 5′-TTTAT GT CGAC GGTGTTGGAAGGGCTACATT-3′, incorporating an EcoRI and SalI site, respectively. RT-PCR was performed using the one-tube method as described by Pfeffer et al. ( 36 ). In brief, 1 μl T24 mRNA (0.5 μg/μl), 10 μM primer (1 μl each), 1.25 U Taq polymerase ( GIBCO BRL ), 100 U SuperScript II RNase H–reverse transcriptase ( GIBCO BRL ), 20 U RNase inhibitor ( Promega Corp. ), 0.25 μl of 10 μM dNTPs, and 2.5 μl 10× PCR buffer containing 500 mM KCl; 100 mM Tris-HCl, pH 8.3; 15 mM MgCl 2 ; and 0.1% gelatin were added to a final total volume of 25 μl, and the PCR steps were programmed using a thermocycler (Eppendorf). The reactions were performed at 50°C for 1 h and followed by 30 cycles at 57°C for 10 s, 72°C for 2 min, and 94°C for 10 s. RT-PCR products were analyzed by agarose gel electrophoresis. For increased expression and purification of recombinant protein, p62 cDNA derived from RT-PCR was subcloned into the EcoRI and SalI sites of pET28a vector, which provides the NH 2 -terminal fusion protein with 6× histidine and T7 epitope tags. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) and purified using nickel column chromatography. The protocol used for the high-level expression and purification of 6× histidine–tagged proteins was performed as described (Qiagen, Inc.). Elution buffer (8 M urea, 0.1 M NaH 2 PO 4 , and 0.01 M Tris, pH 4.5) was used to elute the recombinant protein. The p62 cDNA was transcribed and translated in vitro using TnT-coupled reticulocyte lysate system ( Promega Corp. ) in the presence of [ 35 S]methionine (ICN) as described ( Promega Biotec). Labeled products were used as substrates for immunoprecipitation analysis. Recombinant protein was electrophoresed on 15% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were cut into strips and the recombinant protein bands confirmed by Western blotting. Nitrocellulose strips were incubated with diluted serum at 1:100, and unbound antibodies were removed by washing with PBS containing 0.5% Tween-20 before elution of bound antibodies with 0.5 ml elution buffer (200 mM KH 2 PO 4 , 150 mM NaCl, and 0.1% BSA, pH 2.5). Affinity-purified antibodies were immediately neutralized by the addition of 1 M Tris-HCl, pH 8.7. The antibodies were concentrated with Centricon-30 microconcentrators (Amicon Corp.), and different dilutions (1:5, 1:25, and 1:50) were used for immunofluorescence assay and Western blotting analysis. Four female New Zealand White rabbits were immunized by subcutaneous injections of 0.5 mg of p62 recombinant protein in complete Freund's adjuvant. Rabbits were boosted two times with 0.5 mg p62 recombinant protein in incomplete Freund's adjuvant at 1-mo intervals, and blood was collected 10 d after the last booster injection. Nylon membranes blotted with poly A + RNA isolated from multiple human tissues and several human cancer cell lines were obtained from Clontech . An antisense riboprobe was generated from a 480-bp fragment corresponding to the NH 2 -terminal domain of p62 and labeled with [α- 32 P]UTP ( Clontech ) as described. In brief, the membranes were hybridized with 32 P-labeled p62 riboprobe for 2 h at 74°C, washed in 2× SSC and 0.1% SDS at 74°C for 20 min and in 0.1× SSC and 0.1% SDS at 65°C for 20 min, and exposed to x-ray film for 4 h at −70°C. A 2.0-kb human β-actin cDNA provided by Clontech was used as control probe. Standard protocol for ELISA was used as described by Rubin ( 37 ). Purified p62 recombinant proteins were diluted in PBS to a final concentration of 1 μg/ml for coating Immulon 2 microtiter plates (Dynatech Laboratories). Human sera diluted 1:100 were incubated in the antigen-coated wells. Horseradish peroxidase–conjugated goat anti–human IgG (Caltag Laboratories) and the substrate 2.2′-azinobis (3-ethylbenzthiazoline sulfonic acid; Boehringer Mannheim ) were used as detecting reagents. Each sample was tested in duplicate, and the average OD at 490 nm was used for data analysis. The cutoff value designating positive reaction was the mean OD of normal sera + 3 SD. Initial studies focused on the analysis of serum specimens from 95 HCC patients from Henan Province. Many of these sera contained antibodies reactive with a 62-kD cellular antigen that was present in low concentration in MOLT-4 cell extracts but in higher concentration in extracts from other cell lines. Fig. 1 shows Western blotting of three such sera against whole cell extracts from MOLT-4, T24, and HepG2. Lanes 1, 5, and 9 are normal human serum showing negative reactions. Lanes 2, 6, and 10 show the reactivities of HCC serum YZ. This serum showed strong reactivity with a 90-kD band in MOLT-4 cell extracts and weak reaction with a 62-kD band. With T24 cell extract, the 62-kD band was slightly stronger, but in HepG2 cell extract the 62-kD reactivity was as strong or stronger than the 90-kD reactivity. In addition, a good signal was observed at 50 kD. Serum YL from a different patient contained antibodies primarily against the 90-kD antigen and displayed very weak reactivity with the 62-kD antigen (lanes 3, 7, and 11). A third serum, CH, appeared to have antibodies primarily against the 62- and 50-kD antigens (lanes 4, 8, and 12). In addition, serum CH, as well as YZ, demonstrated weak reactivity with other antigens of higher and lower molecular sizes, but the predominant reactivities detected were against the 90-, 62-, and 50-kD proteins. These data demonstrated that different cell lines showed different levels of expression of these intracellular proteins, with T24 expressing higher levels of the 62-kD and HepG2 expressing higher levels of both 62- and 50-kD antigens than MOLT-4. The above data suggested that in order to isolate cDNA clones encoding the 62-kD antigen, T24 or HepG2 cDNA expression libraries would be the preferred cell lines. HCC serum YZ was used to immunoscreen a T24 λzap cDNA expression library. One positive clone was isolated from 3.0 × 10 5 recombinant plaques, purified to homogeneity, and subcloned in vivo into pBK-CMV. This clone, designated JY1, was a cDNA of 3.3 kb and was shown to have an apparent coding sequence with a termination codon and a long 3′ UTR with a poly A tail. 5′-RACE methodology was used to obtain overlapping 5′ clones using the Marathon-Ready cDNA repertoire derived from human colorectal adenocarcinoma SW480 cell line. Two overlapping independent clones were obtained and analyzed, with the longest clone, H27, containing a cDNA insert of 374 bp. An in-frame TGA stop codon at position 320 bp upstream of a methionine start codon was found. The presumptive full length cDNA is shown in Fig. 2 C. The cDNA has a 5′-UTR of 435 bp, an ORF of 1,668 bp, and a 3′-UTR of 1,564 bp. RT-PCR was performed using T24 cell line RNA as template with one pair of designed primers . The RT-PCR products were of ∼1.7 kb, compatible with the size of the ORF in the isolated cDNA clone . The ORF codes for 556 amino acids with a predicted molecular mass of 62 kD and a calculated pI of 8.59. The 62-kD protein contained two types of RNA binding motifs, the consensus sequence RNA–binding domain (CS-RBD) and four hnRNP K homology (KH) domains ( 38 ). The CS-RBD domain was located in the NH 2 -terminal region, and the four KH domains extended from the middle region to the COOH-terminal region . Three proteins were found to have high degrees of homology to p62 at both nucleotide and protein levels. Table I compares percent similarity and identity of p62 with the other three proteins. KH domain–containing protein overexpressed in cancer (Koc), a putative oncogene ( 39 ), had 66.5% identity and 80.3% similarity to p62. Zipcode binding protein (ZBP1), a β-actin mRNA-binding protein in chicken ( 40 ), had a 70.5% identity and 83.9% similarity to p62, and B3 ( X. laevis TFIIIA–binding protein), an oocyte factor that binds to a developmentally regulated cis element in the TFIIIA gene ( 41 ), showed 69.7% identity and 82.7% similarity to p62. Table I also shows that other KH domain–containing proteins such as FMR1 ( 42 ), hnRNP K ( 43 ), and hnRNP X ( 44 ) showed much lower levels of homology. The sequences of p62, Koc, ZBP1, and B3 are shown in Fig. 3 A, demonstrating the CS-RBD and four hnRNP K homology domains. In addition, a nine–amino acid sequence (VGAIIGKE/KG) of unknown function previously reported in ZBP1 ( 40 ) was also found in the first three KH domains of the other three proteins. A potential REV-like nuclear export signal also found in ZBP1 protein ( 40 ) was present in position 308–319 of p62. The sequence alignments of the four proteins are depicted in Fig. 3 A, and their domain structures are shown in Fig. 3 B. The p62 recombinant protein was expressed using the pET28a vector in E. coli and purified using nickel affinity column chromatography. A 62-kD polypeptide was detected by Coomassie blue staining together with one prominent lower molecular mass product . HCC sera containing antibodies to the 62-kD cellular antigen reacted strongly with the 62-kD recombinant protein and also with the lower molecular weight product . In contrast, HCC sera containing only 90-kD autoantibodies such as serum YL and normal human serum were nonreactive. In vitro–translated products of p62 cDNA showed that the polypeptide migrated at 62-kD and was immunoprecipitated by human anti-p62 prototype serum but not normal human serum . The in vitro–translated products comigrated with the 62-kD antigen detected by Western blotting of HepG2 cell extract . To further confirm that the recombinant protein was identical to the 62-kD cellular protein, four female New Zealand White rabbits were immunized with p62 recombinant protein. Rabbit immune sera immunoprecipitated in vitro–translated products . In addition, Fig. 5 B shows that the cellular 62-kD protein was immunoprecipitated by immune rabbit serum (lane 10) in a fashion similar to human anti-p62 prototype sera (lanes 7 and 8), using extracts from [ 35 S]methionine–labeled HeLa cells as the antigen preparation. Immune rabbit sera were also reactive in Western blotting with 62-kD proteins from T24, HepG2, and A549 cells (data not shown). Fig. 5 B shows that other proteins were also immunoprecipitated, but many of these were also immunoprecipitated by preimmune rabbit serum and normal human sera and are presumed to be nonspecific precipitates. It had been previously observed by immunofluorescent histochemistry that HCC sera with antibodies to the 62-kD antigen were negative for staining of the nucleus but positive for cytoplasmic staining. Immune rabbit antiserum affinity-purified from recombinant protein showed cytoplasmic staining, as depicted in Fig. 6 B. The pattern of human HCC prototype serum YZ is shown in Fig. 6 A. The fine details of cytoplasmic staining of human serum YZ and rabbit immune sera are somewhat different, with rabbit serum showing a coarser pattern of cytoplasmic staining than human serum. This could be related to the fact that human sera are polyclonal and might contain other autoantibodies. The relevant finding was that both human HCC sera with anti–p62-kD antigen reactivity and rabbit immune sera were reactive with antigens that were cytoplasmic in location. Using a 480-bp fragment corresponding to the COOH-terminal domain of p62 as probe , two major forms of p62 transcripts were detected in Northern blotting using commercially available poly A + RNA from a number of human tissues and cell lines. Fig. 7 demonstrates that there was a 3.7-kb transcript (lower arrow) that was reactive with the probe, as well as a 5.2-kb transcript (upper arrow). The 3.7-kb transcript of p62 was found in heart and placenta, whereas brain, lung, liver, kidney and pancreas gave much lower signals or were negative for p62 expression. A signal for skeletal muscle migrated somewhat more slowly than the 3.7-kb transcript (see data showing the probe corresponding to the NH 2 -terminal domain below). High levels of the 3.7-kb transcript were also detected in HeLa, K-562 (chronic myelogenous leukemia), SW480 (colorectal adenocarcinoma), A549 (lung carcinoma), and G361 (melanoma) cell lines, whereas low expression was observed in HL60 (promyelocytic leukemia), MOLT-4 (lymphoblastic leukemia), and Raji (Burkitt's lymphoma) cell lines. The presence of the 3.7-kb transcript in these cell lines showed good correlation with the presence of a 62-kD polypeptide antigen in extracts of these cell lines, whereas cell lines negative for the 3.7-kb transcript contained negligible or barely detectable amounts of 62-kD polypeptide. The data are not shown for all cell lines, but as can be seen in Fig. 1 , MOLT-4 cell extracts contained low levels of p62 antigen and low levels of 3.7-kb transcript . The Northern blot data just described were confirmed with an antisense riboprobe generated from a 687-bp fragment corresponding to the NH 2 -terminal domain of p62, and the same membrane shown in Fig. 7 A was stripped and rehybridized with this probe. The 3.7- and 5.2-kb transcripts were specifically detected by this probe, a result similar to the observations shown in Fig. 7 A with the exception that the “slower” 3.7-kb band in skeletal muscle was not detected. From these observations, we propose that both the 3.7- and 5.2-kb transcripts represent cellular mRNAs encoding the 62-kD protein. The identity of the 5.2-kb transcript has not been determined, but it could represent an alternative mRNA for the 62-kD protein with extra 5′ upstream or 3′ untranslated sequences. An enzyme-linked immunosorbent assay system was developed using p62 recombinant protein as antigen and a total of 242 sera from humans with different conditions were examined for reactivity. Table II shows that detectable antibodies to p62 antigen were present in 21.1% of patients in a group of patients with HCC from Henan Province. However, in sera from patients with conditions that are known to be precursor diseases to HCC, including asymptomatic HBsAg, acute hepatitis, and chronic hepatitis, no antibodies to p62 were detected. Normal human sera from patients who came from Henan Province or the San Diego area were also negative for p62 antibodies. Study of human tumor antigens recognized by the autologous host has had a long history, and T cell–recognized epitopes on human tumor cells have been extensively characterized ( 30 ). However, antibody-defined tumor antigens have been receiving greater attention recently, and several centers have used a method called SEREX (serological analysis of recombinant cDNA expression libraries), libraries of cDNA constructs made from mRNA extracted from tumor tissue ( 29 ) and screening of such libraries with autologous serum. From these studies, several novel as well as previously defined tumor antigens have been identified ( 30 ). Our approach was not restricted to use of cDNA libraries from autologous tumors because our previous studies had shown that antibodies in HCC sera were reactive with antigens expressed by a variety of tissue culture cell lines ( 23 – 25 ). The autoantigens identified included some previously recognized in autoimmune diseases such as lupus and scleroderma ( 22 , 23 ). However, an unusual feature in the HCC model was that novel antigen-antibody reactions were detected in some patients during transformation from chronic liver disease to malignancy. Therefore, use of such sera to isolate and identify antigens might reveal special signatures of cancer cells. A novel protein with alternative splicing factor motifs ( 25 ) and a cell cycle–related nuclear protein with WD-40 motifs ( 26 ) have been isolated. These novel autoantigens, including p62 reported here, are not restricted to cancer cells but are also expressed in normal cells, an observation which has also been reported for cancer antigens in melanoma. These antigens have been called differentiation antigens ( 28 ). At this point, it is unclear why some differentiation antigens become capable of provoking autoimmune responses, but possibilities which have been raised in the case of p53 include gene mutations, gene product overexpression, and unusual complexes with other cellular proteins such as heat shock proteins ( 27 , 45 ). In addition to the remarkably high percentage of similarity and identity between p62 on the one hand and ZBP1 and Koc on the other, there are several features of these proteins that are of interest. ZBP1 is a chicken protein of 68 kD which was identified through its property of binding to a conserved element in the 3′ untranslated region of β-actin mRNA ( 40 ). Rabbit antibodies raised against ZBP1 polypeptides were shown to bind to β-actin mRNA in the leading edge of the lamella in cells such as chicken embryo fibroblasts and 3T3 fibroblasts ( 40 ). Studies are in progress to determine whether recombinant p62 is capable of binding in vitro to the “zipcode” element of β-actin mRNA as is the case with ZBP1. The Koc protein was isolated following analysis of differential gene expression in pancreatic cancer tissue ( 39 ). High transcript levels of Koc were found not only in pancreatic cancer tissue but also in soft tissue sarcoma, gastric cancer, and colon cancer. Differences in mRNA expression of Koc in different tissues has been reported ( 39 ). We have also observed different transcript levels of p62 in different tissues and, in addition, high levels of expression of p62 transcript in some cell lines (HeLa, K-562, SW480, A549, and G361) but low expression in others including the promyelocytic leukemia HL-60, lymphoblastic leukemia MOLT-4, and Burkitt's lymphoma Raji. It is perhaps of some interest that the two human members (p62 and Koc) of this putative family of RNA-binding proteins appear to be associated in some way with cancer. It has recently been reported that two different Koc-homologous genes were also identified by SEREX methodology ( 46 ), but sequence information concerning these genes and their relationship to cancer are not yet available. Previously, we reported that several patients with HCC mount de novo immune responses to nuclear antigens at the time of conversion from chronic hepatitis or liver cirrhosis to HCC ( 24 ) and that the autoantibodies that were produced against intracellular antigens might be regarded as immune system reporters of abnormal intracellular molecular events. Novel proteins that have been detected include HCC1 ( 25 ), a putative member of the SR family of alternative splicing factors, and SG2NA, a protein highly expressed in the S and G2 phases of the cell cycle ( 26 ). p62 appears to be another such molecule, but unlike the antibody responses to HCC1 and SG2NA, which were observed in individual patients, the autoimmune response to p62 was detected in >20% of one group of HCC patients, suggesting that a shared stimulus might be inciting the immune responses. This shared stimulus could be environmental in nature, but this is as yet only conjecture. An important study that could not be performed at this time was the analysis of HCC tissues to determine whether there were abnormalities in the p62 gene or in its expression, as tissue specimens were not available in this retrospective study. This will be the focus of a prospective study in newly identified HCC patients with autoantibodies to p62. | Study | biomedical | en | 0.999996 |
10190902 | The LACK 156–173 peptide (ICFSPSLEHPIVVSGSWD), LACK-N164 and LACK-K164 mutant peptides with the designated alterations of H at position 164, and the OVA 323–336 peptide (ISQAVHAAHAEINE) were synthesized on an Advanced Chemtech Multiple Peptide Synthesizer. Peptides were purified by reverse phase HPLC and their identities confirmed by analysis with an LCQ mass spectrometer (Finnigan MAT). The full length rLACK sequence was ligated downstream of a hemagglutinin epitope, six histidine residues, and factor X cleavage site in the pET3a-δ9 vector as described ( 5 ). A 41–amino acid deletion construct (rLACKΔ41) that excised the LACK 156–173 epitope was created and expressed from the same vector as described ( 5 ). rLACK-N164 and rLACK-K164 were produced by site-directed mutagenesis of the nucleotide triplet at bp 684–686 from CAC (H) to AAC (N) or AAG (K), respectively, based on codon usage in Leishmania ( 9 ). The XbaI/HindIII fragment of rLACK (1,134 bp) was cloned into the pGEM11Zf(−) vector ( Promega ). Mutations were generated with the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) using the primers 5′-CGTCGCTGGAGAACCCGATCGTG-3′ (N) or 5′-CGTCGCTGGAGAAGCCGATCGTG-3′ (K) according to the manufacturer's protocol. The mutated rLACK proteins were sequenced to confirm their identities and cloned using the XbaI and HindIII sites into pET3a-δ9. Escherichia coli BL21 (DE3)plysS (Novagen) were transformed, and the expressed proteins were purified using [Ni]nitrilotriacetic acid chromatography as described ( 5 ). Female BALB/c, C57BL/6 (The Jackson Laboratory or IFFA Credo), and B10.D2 mice (The Jackson Laboratory ) were housed in the University of California San Francisco or University of Lausanne pathogen-free animal facilities and used at 8–10 wk of age. Designated mice were thymectomized at 5 wk of age using standard methods. LACK T cell receptor– specific transgenic (ABLE) mice are TCR-transgenic mice that express a Vβ4/Vα8 TCR that recognizes a peptide epitope comprising amino acids 156–173 from the Leishmania LACK antigen in the context of MHC class II I-A d molecules. The generation and characterization of the ABLE mice are described elsewhere ( 4 ). ABLE mice were backcrossed 10 generations onto the BALB/c background and, where indicated, onto backcrossed BALB/c TCR constant region α chain deletion mutants (TCR-Cα 0 ; 10) to create BALB/c ABLE TCR-Cα 0 mice as described ( 4 ). L. major strains WHOM/IR/−/173 (designated IR/173) and MRHO/Sv/59/P (designated LV39 were passaged and maintained as described ( 4 , 5 ). Groups of 4–10 mice were infected in the hind footpads with purified metacyclic (4 × 10 5 ) or stationary phase (2 × 10 6 ) promastigotes as previously described ( 4 , 5 , 11 ). After inoculation, disease progression was monitored using a metric caliper to quantitate footpad size. Animals were killed at the designated times, and the popliteal lymph nodes were harvested for the evaluation of cell types and cytokine production as described ( 5 ). Serum was collected terminally for quantitation of IgE by ELISA as described ( 11 ), and the footpads and spleens were used to quantitate the parasite burden by limiting dilution ( 12 ). I-A d molecules were affinity purified from cell lysates of A20 lymphoma cells using anti–I-A d mAb MKD-6 (American Type Culture Collection [ATCC]). Peptides were tested for binding to I-A d as measured by their capacity to inhibit the binding of 125 I-radiolabeled OVA 323–336 as previously described ( 13 ). Spleen and lymph nodes were harvested from BALB/c ABLE TCR-Cα 0 mice and used to produce single-cell suspensions after disruption through a 0.75-μm nylon mesh filter. Cells were washed, the red cells were lysed, and the resulting populations of ABLE T cells and APC were distributed to duplicate wells of round-bottom microtiter plates (10 6 cells/well) in cell culture medium with the indicated concentrations of synthetic peptides in a final volume of 0.2 ml. Supernatants were harvested after 48 h for cytokine analysis by ELISA. The wells were pulsed at 48 h with 1 μCi [ 3 H]thymidine, and cell proliferation was assessed 18 h later. CD4 + T cells from BALB/c ABLE TCR-Cα 0 mice were enriched from spleen and lymph node cell suspensions by antibody- and complement-mediated lysis of B cells, MHC class II-, and CD8-expressing cells using mAbs J11d, BP107, and 3155 (ATCC), respectively, and low-toxicity rabbit and guinea pig complement (Cedarlane Labs., Ltd.). The resulting populations were 80% Vβ4 + cells, of which 35–40% were CD4 + and the remainder CD4 − CD8 − as previously described ( 14 ). Irradiated spleen cell populations from BALB/c TCR-Cα 0 mice were used as APC. Antagonism was quantitated using the method of DeMagistris et al. ( 13 ), with slight modifications. APC (10 7 cells/ml) were prepulsed with suboptimal concentrations of the wild-type LACK peptide (0.008–0.2 μM as established in preliminary experiments) in culture medium for 2 h at 37°C. The APC were washed, irradiated, and distributed to 96-well round-bottom microtiter plates (2 × 10 6 cells/well) and further incubated with varying concentrations of the designated peptides (0.01–100 μM) for 2 h at 37°C. The plates were washed, and enriched ABLE T cells were added using 1.6 × 10 5 T cells/well in 0.2 ml medium. After 48 h, the supernatants were collected and analyzed for cytokines by ELISA. Proliferation was assessed at hour 66, after pulsing for the final 18 h with 1 μCi [ 3 H]thymidine/well. Groups of BALB/c mice were injected in the hind footpads with 5, 25, or 50 μg of the designated rLACK protein or 25 μg chicken egg OVA in 50 μl buffer. At various time points, the popliteal lymph nodes were harvested and mRNA purified for analysis of IL-4 transcripts using a semiquantitative reverse transcriptase (RT)-PCR assay as previously described ( 15 ). Groups of these treated control or adult thymectomized mice were challenged either 24 h or 10, 20, or 30 d later with either designated rLACK proteins or viable L. major and analyzed by similar methods. BALB/c ABLE-Cα 0 mice were immunized in both footpads with 25 μg of the purified rLACK proteins or chicken egg OVA in 50 μl buffer and, 24 h later, the popliteal lymph nodes were collected and single-cell suspensions prepared. Cells (2 × 10 5 ) were analyzed by flow cytometry (FACSVantage™; Becton Dickinson ) after incubation with a combination of fluorescein isothiocyanate–conjugated anti-Vβ4, PE-conjugated anti-CD4, and biotinylated anti-CD69 mAbs, followed by streptavidin-tricolor (all from Caltag Labs.). Nonthymectomized or adult thymectomized BALB/c mice were immunized in the hind footpad with 25 μg purified rLACK proteins or chicken egg OVA in 50 μl of 50 mM Tris/100 mM NaCl, pH 8.0. Mice were infected 24 h later with the designated strains of L. major promastigotes in the left footpad and the course of infection monitored as described above. IL-4 and IFN-γ were measured using sandwich ELISA with mAbs 11B11 and biotinylated BVD6 for IL-4 detection and R46A2 and biotinylated XMG1.2 for IFN-γ detection as described ( 11 ). Samples were normalized to standard recombinant controls. The limits of detection in these assays were 50 pg/ml for IL-4 and 1 ng/ml for IFN-γ. Cytokine production by individual lymphocytes was assessed by ELISPOT assay as described ( 11 ). Total serum IgE was measured using ELISA with mAbs B.IE.3 and biotinylated EM-95 and normalized to concurrently analyzed standards ( 11 ). Cytokine mRNA transcript abundance was quantitated using RT-PCR with the competitor plasmid pPQRS as described ( 5 , 15 ). In brief, cDNA samples were first normalized for expression of a constitutively expressed gene, hypoxanthine phosphoribosyltransferase (HPRT), and then quantitated for expression of IL-4 and IFN-γ as compared with competitor pseudogene transcripts amplified within the same reaction. The ratio of the authentic and competitive amplicons was quantitated using densitometry. Using overlapping synthetic peptides and a panel of T cell hybridomas generated from BALB/c mice immunized with the recombinant protein, a single I-A d – restricted epitope in LACK was localized to amino acids 156–173, comprising the sequence ICFSPSLEHPIVVSGSWD (data not shown). Almost all hybridomas reactive to LACK expressed a Vβ4/Vα8 heterodimeric TCR, although considerable junctional diversity was apparent. The putative CDR3 peptide contact domain, however, was generally conserved in length and charge, with a negatively charged QE or QD motif in the TCR β chain of each of the LACK-reactive hybridomas ( 3 ). Similarly, hybridomas established from the lymph node cells of infected BALB/c mice that expressed the Vβ4 TCR contained the QE motif in the CDR3; one had a charged WD motif at the same position ( 2 ). Such features suggested that a positively charged amino acid within the LACK antigenic determinant represented a critical TCR contact residue. Based on the use of histidine and other charged residues at TCR contact points among peptides binding to I-A d ( 16 ), we mutated the histidine at position 164 in the wild-type peptide (LACK) to asparagine or lysine, thus creating peptides LACK-N164 and LACK-K164, respectively. The relative affinities for MHC class II molecules by LACK and the LACK analogues were tested by assaying their capacities to compete with an I-A d ligand of known affinity, chicken egg OVA peptide 323–336 . By this assay, each of the LACK-derived peptides displayed binding affinities for I-A d in the same nanomolar range as the OVA 323–336 reference peptide; if anything, they showed slightly stronger affinities (Table I ). Substitution of H164 in the wild-type LACK determinant by N or K did not, therefore, affect its binding affinity for MHC class II molecules. ABLE mice express a transgenic TCR derived from a LACK-reactive Vβ4/Vα8 T cell clone that is activated by the LACK 156–173 peptide in the context of I-A d ( 4 ). These mice have been crossed to BALB/c TCR-Cα 0 mice, thus creating BALB/c ABLE TCR-Cα 0 mice. These mice express a monoclonal αβ T cell repertoire consisting exclusively of the LACK-reactive TCR transgene and were used as a source of T cells, designated ABLE T cells. ABLE T cells proliferated in response to the LACK wild-type peptide at low concentrations (7 nM) but not after stimulation with the LACK-N164 or LACK-K164 analogues, even at concentrations up to 4 μM . Although ABLE T cells generated both IFN-γ and IL-4 in culture supernatants after incubation with the LACK peptide, neither cytokine was detected after incubation with the two analogue peptides nor with the irrelevant OVA peptide that also binds I-A d . Thus, a single amino acid substitution at position 164 in the LACK T cell epitope substantially altered reactivity of the transgenic T cells, indicating that this amino acid position is likely to be a critical TCR contact residue. The LACK-derived peptides were next analyzed in an antagonism assay that was developed to avoid peptide competition at the level of MHC occupancy and thus allow measurements of events mediated by the TCR ( 13 ). Spleen cells from BALB/c TCR-Cα 0 mice were used as APC and were preincubated with a suboptimal concentration (0.2 μM) of the wild-type LACK peptide. Washed and irradiated APC were then incubated with increasing concentrations (0.1–100 μM) of the LACK analogue peptides or the OVA 323–336 peptide before ABLE T cells were added and assessed for their capacity to proliferate and produce cytokines. LACK-K164 showed dose-dependent inhibition of proliferation to the wild-type peptide; 50% inhibition occurred at a concentration of 10 μM of the analogue peptide . At similar concentrations, the peptide also inhibited IL-4 and IFN-γ production. LACK-N164 inhibited the proliferation of ABLE T cells only at very high concentrations (>100 μM). The production of IL-4 was inhibited comparably to the LACK-K164 peptide, but IFN-γ production was inhibited consistently less by LACK-N164 in multiple assays. The unrelated OVA 323–336 peptide displayed no inhibitory activity. Thus, in the presence of otherwise stimulatory amounts of the wild-type LACK peptide, the two analogue peptides behaved as TCR antagonists. Of the TCR-mediated functions tested, LACK-N164 preferentially inhibited IL-4 production by ABLE T cells, whereas the capacity to proliferate and produce IFN-γ was less affected; LACK-K164 was more global in its inhibitory capacities. The same amino acid substitutions were introduced into the full length rLACK protein by site-directed mutagenesis, creating rLACK-N164 and rLACK-K164 altered proteins. When tested in vitro for its capacity to stimulate ABLE T cells, the rLACK protein stimulated proliferation and IL-4 and IFN-γ production at molar concentrations comparable to those of the wild-type LACK peptide. In contrast, the altered rLACK proteins, as their peptide counterparts, did not stimulate proliferation or measurable cytokine production over a wide range of concentrations (data not shown). To assess the activity of the rLACK proteins in vivo, ABLE-Cα 0 mice were injected in the hind footpads with 25 μg of purified rLACK, rLACK-N164, rLACK-K164, or OVA. After 24 h, the popliteal lymph node cells were recovered and analyzed using flow cytometry for activation, as assessed by expression of CD69 and enlargement by light-scattering characteristics. Inoculation of rLACK effectively targeted the transgenic T cells: 80% of Vβ4 + cells expressed CD69 and forward/side scattering increased significantly (data not shown). The total number of transgenic T cells actually decreased in the draining lymph nodes (from 2.9 × 10 5 after OVA to 1.3 × 10 5 after rLACK), consistent with antigen-mediated deletion as previously described in other TCR-transgenic mice ( 17 , 18 ). In contrast, Vβ4 + T cells collected from animals injected with the rLACK analogues showed CD69 induction and forward/side scattering indices that were only modestly greater than those from cells collected from animals injected with the control protein, OVA . Furthermore, the total number of transgenic T cells in these mice was not significantly different from the number of transgenic T cells in mice that received OVA (data not shown). Thus, as assessed by these criteria, the rLACK analogues did not activate ABLE T cells in vivo in a manner comparable to the cognate LACK protein containing the wild-type epitope. Prior experiments demonstrated that the LACK antigen induced prominent IL-4 expression in Vβ4/Vα8 CD4 + T cells after injection into BALB/c mice that reached levels 30–100-fold greater than after injection of a construct with the I-A d epitope deleted ( 5 ). Over a 5–50 μg range of rLACK, IL-4 mRNA was induced 100-fold, whereas no IL-4 mRNA was induced by either the 41–amino acid LACK deletion mutant or rLACK-K164 . Although a 10-fold induction of IL-4 mRNA was seen after injection of 5 μg rLACK-N164, no IL-4 mRNA was induced after injection of 25- or 50-μg doses. None of the LACK derivatives caused induction of IFN-γ mRNA under the conditions used. After immunization with CFA, however, each of the constructs, rLACK, rLACK-K164, and rLACK-N164, was capable of inducing a proliferative response from subsequently isolated popliteal lymph node CD4 + T cells in response to their respective LACK 156–173 epitopes (stimulation indices increased 10–18-fold; data not shown). No proliferation was induced by any of the LACK epitopes after immunization with the LACK deletion mutant or OVA. The 25-μg protein dose was selected for use in subsequent experiments. To assess the capacity to alter the response of the endogenous LACK-reactive repertoire, mice were first injected with 25 μg of the LACK analogue proteins and then, 24 h later, with either the authentic LACK protein or viable L. major promastigotes, both shown previously to activate a brisk IL-4 mRNA response in BALB/c CD4 + T cells ( 5 ). In three separate experiments, prior injection of either analogue protein substantially decreased the subsequent activation of the IL-4 response, consistent with an alteration of the normal Vβ4/Vα8 CD4 + T cell response . Prior injection of the LACK construct with the deleted I-A d epitope or an unrelated I-A d epitope (OVA) did not affect the subsequent IL-4 response. Kinetic analysis, in which the second injection of recombinant LACK was delayed 10, 20, or 30 d after the initial immunization, revealed that IL-4 nonresponsiveness was maintained for 10–20 d in mice that had been injected with the mutated LACK analogues but then subsequently recovered. Recovery of IL-4 responsiveness to LACK or L. major injection was ablated by prior thymectomy (data not shown). Based on the capacity of LACK analogue proteins to abrogate the early IL-4 response in BALB/c mice, we assessed the ability of immunization to render these mice resistant to progressive disease. Cohorts of mice were immunized once with 25 μg of the various recombinant proteins in the footpad and challenged 24 h later with a lethal infectious dose of wild-type L. major promastigotes of either the IR/173 or LV39 strain. In three separate experiments with the IR/173 parasite, the course of disease was significantly attenuated in animals that received rLACK-N164; no attenuation was seen in animals that received rLACK-K164 or any of the control proteins, including the wild-type LACK protein . Whereas animals in all of the other groups had to be killed by week 8, mice that received rLACK-N164 controlled disease up to 12 wk after inoculation, when the experiment was terminated. Parasitologic control was confirmed by limiting dilution of tissues that demonstrated a 2–4-log reduction in parasite numbers. Immunologic analysis revealed a threefold reduction in the number of IL-4–producing cells in the draining lymph nodes and in serum IgE levels, whereas the number of IFN-γ–producing cells was similar in all groups. Protection was more dramatic using the LV39 strain. Similar to the results using the IR/173 strain, rLACK-N164 but not rLACK-K164 provided lasting protection in a subgroup of nonthymectomized mice . The LACK protein itself provided protection in approximately half of both wild-type and thymectomized BALB/c mice . Strikingly, either of the altered LACK proteins, in contrast to the LACK deletion mutant or OVA controls, provided a complete protection in thymectomized mice that was sustained over 100 d . When studied at the conclusion of these experiments, the cure phenotype was associated with attenuation of IL-4 production and control of parasite growth in the footpads that was entirely concordant with the lesion phenotype (data not shown). L. major includes a heterogeneous group of protozoa strains that express some 10,000 proteins from a 35.5-megabase genome ( 19 ). Despite this complexity, the early immune response is highly focused on a single epitope from the parasite LACK antigen in mice that express I-A d MHC class II molecules. As shown here, targeting T cells that recognize this epitope using ligands that differed by a single amino acid from the natural epitope was capable of redirecting an otherwise ineffective immune response with a fatal outcome to a completely protective response with long-term cure. The specificity of the immune intervention suggests limited plasticity in the innate LACK-reactive repertoire in I-A d –bearing mice, as well as limited ability of Leishmania parasites to mediate progressive infection in such mice in the absence of exuberant LACK recognition. The mechanisms underlying the dominant recognition of the LACK epitope remain unclear. Recognition does not seem related simply to the abundance of the LACK protein. As compared with other parasite molecules like the major surface protease gp 63 or the major surface glycolipid LPG, which are present in ∼5 × 10 5 and 3–5 × 10 6 molecules per organism ( 20 , 21 ), respectively, LACK was less abundantly expressed. Quantitation against recombinant standards showed that LACK comprised only ∼0.03% of total cellular protein or ∼30,000 molecules per organism (Pingel, S., and R. Locksley, unpublished data). The LACK epitope displayed in vitro affinity for I-A d that was comparable with endogenously eluted I-A d peptides ( 22 ) and contained a centrally disposed histidine residue, creating a charged element that has been noted in other peptides that bind this MHC molecule. Presumably, the dominance of the epitope must result from some confluence of stability and processing of the peptide, the efficiency of targeting to MHC class II molecules, and/or the size of the responding T cell repertoire ( 23 ). Equally perplexing is the dominant nature of the Vβ4/Vα8 TCR response to the I-A d /LACK peptide complex. The convergence of the immune response on the LACK epitope through use of a dominant Vα/Vβ-paired TCR has been reported using other immunogenic peptides ( 24 ), suggesting that other antigens or adjuvant-like molecules from live parasites do not affect this clonal affinity maturation process. Earlier studies reported the ability to vaccinate susceptible BALB/c mice against L. major using LACK antigen administered in a manner such that LACK-specific Th1 cells were generated ( 3 , 25 ). Indeed, LACK-specific Th1 cells were alone sufficient to establish substantial control over infection with L. major , demonstrating that immunoreactive LACK peptide is expressed in vivo at physiologically important levels ( 4 ). Despite the capacity of LACK-specific T cells to control infection with the parasite in vivo, such T cells are not required. Thus, BALB/c mice rendered deficient in CD4 + T cells that recognize this dominant epitope, either through thymic expression and central deletion or by superantigen-mediated deletion of all Vβ4 + CD4 + T cells, were capable of containing L. major infection ( 3 , 5 ). These experiments suggested that LACK recognition was required for establishing the susceptible state of BALB/c mice, although neither method directly targeted epitope-specific T cells. Thus, overexpression of LACK antigen in the thymus might affect the T cell repertoire in ways other than deletion of LACK-reactive T cells. Similarly, deletion of all Vβ4 + CD4 + T cells targets cells of additional specificities but unknown contributions to defense against Leishmania . The ability of peptide ligands that differed at a single amino acid residue to affect the subsequent course of disease in susceptible BALB/c mice argues strongly for highly conserved specificity to the Th2-mediating repertoire. Altered ligands are presumed to anergize or functionally alter discrete populations of T cells by their ability to establish incomplete signaling through the TCR complex (reviewed in 26 ). Modulation of cytokine patterns by altered ligands has been previously demonstrated ( 27 ), although application of this technology to an acute infectious process has been infrequently examined. Redirection of Th subset differentiation, or immune deviation, has been reported by either variations in antigen dose ( 28 , 29 ) or through use of altered peptide ligands ( 30 ). For a variety of reasons, we consider it unlikely that immune deviation can account for the protection mediated by the altered LACK proteins. First, over a wide dose range, the LACK peptide caused no shift in the production of IL-4 and IFN-γ relative to each other by the TCR-transgenic ABLE T cells. Second, the altered LACK peptides induced neither proliferation nor cytokine production by ABLE T cells. Third, the massive activation of ABLE T cells after injection of LACK was absent after injection of the altered LACK antigens. Finally, injections of lower doses of LACK or the altered LACK proteins into BALB/c mice did not induce early production of IFN-γ, rather than IL-4, mRNA. We could, therefore, find no evidence for the establishment of a LACK-specific Th1 response that could account for the protection mediated by the altered LACK analogues. More likely, protection of susceptible mice was accomplished through tolerance or deletion of LACK-reactive T cells, a mechanism consistent with previous experimental findings ( 5 , 7 ). Both altered LACK proteins antagonized IL-4 production by transgenic T cells in response to LACK. When used to immunize BALB/c mice, they abrogated the early IL-4 response to L. major parasites. Treated mice were able to control parasite multiplication of the LV39 strain up to 5 wk; over prolonged periods, and with both the LV39 and IR/173 strains, mice pretreated with the rLACK-N164 protein demonstrated persistent immunity. Thymectomized BALB/c mice immunized with either rLACK-N164 or rLACK-K164 were completely protected, a finding consistent with the ability of the altered LACK proteins to abrogate IL-4 production by Vβ4/Vα8 CD4 + T cells in these mice. Presumably, the delayed yet progressive disease in nonthymectomized LV39-infected mice was dependent on new thymic emigrants. Immunization with the LACK protein itself conferred protection to some mice. As demonstrated using the TCR-transgenic mice, this presumably relates to the capacity of the cognate ligand to mediate peripheral deletion of high-affinity LACK-specific T cells. The observed differences in the overall grade of protection between the two strains of L. major were surprising, but such differences in virulence have been previously described ( 31 , 32 ), as have subset responses within cohorts of identically treated mice, as seen here among rLACK- and rLACK-N164–treated mice ( 33 – 35 ). Boosting or otherwise optimizing the immunization schedule might have enhanced protection against the IR/173 strain. The rLACK-N164 ligand, however, conferred protection against both L. major strains, suggesting that identification of dominant antigens from pathogens can be used to target disease-producing T cells in a highly sequence-specific manner. Previous studies have documented that different T cell clonotypes can respond to the same antigen within a given T cell repertoire. Furthermore, considerable cross-reactivity is an essential feature of the T cell receptor, which assures that pathogenic peptides are efficiently recognized ( 36 ). Our results suggest that the endogenous LACK-specific repertoire is highly constrained in its CDR3 recognition domain. Limited plasticity of the endogenous T cell repertoire has been previously noted with certain peptide antigens ( 37 ), suggesting that infectious diseases may have contributed to the evolutionary divergence of V region genes. Expression of dominant epitopes by parasites, together with the diversity and size of the responding host T cell repertoire, might greatly affect the outcome of such infections and thus contribute to the highly diverse clinical manifestations of leishmaniasis in human populations. L. major contains two LACK genes expressed in tandem from the same chromosome. Apart from what is known regarding their mammalian homologues, little is known regarding the biochemical action of these proteins. Aside from the potential vaccine use of this antigen ( 3 , 25 ), additional study promises to shed much light on the coevolution of host and parasite within the context of the immune system, MHC recognition, and the T cell repertoire. Such studies may have great implications for our understanding of the basis for susceptibility and resistance to infectious diseases. | Study | biomedical | en | 0.999997 |
10190903 | C57BL/6 mice were purchased from Charles River Japan. Vα14 NKT cell–deficient (Jα281 −/− ) and CD1d −/− mice were established by specific deletion of the Jα281 and CD1d gene segment, respectively ( 3 , 10 ). All mice used in this study were at 5–8 wk of age and were maintained in specific pathogen– free conditions. α-GalCer [(2S,3S,4R)-1- O -(α- d -galactopyranosyl)-2-( N -hexacosanoylamino)-1,3,4-octadecanetriol] used for this study was provided by Dr. Y. Koezuka (Kirin Brewery Co., Ltd., Gunma, Japan ). The stock solution of α-GalCer (220 μg/ml) was diluted in 0.5% polysorbate 20 (Nikko Chemical) in 0.9% NaCl solution. This stock solution was further diluted into an appropriate concentration with saline and used for the experiments. A vehicle control solution was prepared from a solution of 0.5% polysorbate 20 in 0.9% NaCl solution. The vehicle control was used in all experiments. Spleen cells were incubated on nylon wool columns for 45 min, and the nonadherent cells were used for the isolation of NKT cells, NK cells, CD4 + T cells, and CD8 + T cells by cell sorting using a FACS Vantage™ instrument ( Becton Dickinson ). All mAbs used in these experiments (mAbs against NK1.1, CD4, CD8, and TCR-α/β) were purchased from PharMingen . Unless noted otherwise, NK1.1 + TCR-α/β + cells were used as purified NKT cells. The stained cells were isolated using the FACS Vantage™. The purity of the sorted cells was >98%. The details of the staining and sorting have been described previously ( 11 ). DCs were prepared according to the method of Steinman et al. ( 12 ) with some modifications. In brief, spleen cells were incubated in 10-cm plastic dishes (Falcon; Becton Dickinson ) for 2 h, and the nonadherent cells were removed from the culture. The adherent cells were further incubated overnight and the nonadherent cells were harvested. Then, CD11c + B220 − CD4 − CD8 − cells were isolated from the nonadherent populations by cell sorting and used as the source of DCs. Generally, DCs (10 5 ) were cocultured with purified NK1.1 + TCR-α/β + NKT cells (2 × 10 5 ) in the presence of 50 ng/ml of α-GalCer in 96-well U-bottomed plates (Costar Corp.). After incubation for 36 h, the culture supernatants were harvested to detect cytokine levels. IL-4 or IFN-γ activity in culture supernatants was determined using the Biotrac™ mouse IL-4 or Biotrac™ mouse IFN-γ ELISA system (Nycomed Amersham plc ). Serum samples were obtained from C57BL/6 mice 24 h after injection of α-GalCer (200 ng/mouse) and/or IL-12 (200 U/mouse; donated by Genetics Institute, Inc., Cambridge, MA), and cytokine levels were measured using ELISA kits (Nycomed Amersham plc ). IL-12 p70 activity in culture supernatants was measured using Intertest-12X™ ELISA kits ( Genzyme Corp. ). The natural killing activity of spleen cells was determined by 4-h 51 Cr-release assays using YAC-1 cells as target. 1 lytic unit (LU) was defined as the number of effector cells required to cause 25% lysis of 2,500 target cells as described previously ( 13 ). Wild-type C57BL/6 mice received an injection of α-GalCer (200 ng/mouse i.v.), and 6 h later mice were injected with recombinant IL-12 (200 U/mouse i.p.) or saline. 1 d after treatment with IL-12, IFN-γ production in the serum and NK activity of spleen cells were determined. Control mice were treated with vehicle only. C57BL/6, CD1d −/− , and Vα14 NKT cell–deficient mice were injected with α-GalCer (2 μg/mouse i.v.) or vehicle. At different time points (0–6 h) after treatment, mice were killed and spleen cells were isolated. TaqMan™ real-time quantitative reverse transcription (RT)-PCR assay was carried out for the detection of IL-12R mRNA expression by these cells according to published methods ( 14 ). In brief, total RNA extracts from the cells were added to the master mixture. To detect the amount of the IL-12R mRNA RT-PCR amplificon, target (IL-12Rβ1, IL-12Rβ2) and control (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) hybridization probes were mixed with target and control PCR primers, respectively. This mixture was transferred to a set of thermocycler tubes and transcribed at 42°C for 30 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 1.5 min, and analyzed using an ABI PRISM 7700 sequence detector (Applied Biosystems). IL-12Rβ1 and IL-12Rβ2 mRNA expression were estimated from the ratio of fluorescence intensity to GAPDH. IL-12R expression induced by α-GalCer is indicated in the figures as induction index, calculated as follows: induction index = IL-12R expression of α-GalCer–stimulated sample/IL-12R expression of unstimulated sample. TaqMan™ probes used for these analyses are as follows: IL-12Rβ1 mRNA-605T, 5′-CGGATGCCCACAACGAATTGGA-3′; IL-12Rβ2 mRNA-551T, 5′-AGCCACCTCAAAACATATCATGTGTCCAGG-3′; GAPDH-542T, 5′-CCTGGCCAAGGTCATCCATGACAACTTT-3′. PCR primers used for these analyses are as follows: IL-12β1 mRNA, forward primer (-563F) 5′-AATGTGTCTGAAGAGGCCGGT-3′ and reverse primer (-657R) 5′-GAGTTAACCTGAGGTCCGCAGT-3′; IL-12Rβ2 mRNA, forward primer (-529F) 5′-ATCTCAGTTGGTGTTGCTCCA-3′ and reverse primer (-602R) 5′-GCCACAGTTCCATTTTCTCCT-3′; GAPDH, forward primer (-368F) 5′-CTTCACCACCATGGAGAAGGC-3′ and reverse primer (-605R) 5′-GGCATGGACTGTGGTCATGAG-3′. Wild-type C57BL/6 mice were injected with 500 μg i.p. anti–IFN-γ mAb (R4-6A2; PharMingen ) or IL-12 (C15.1 and C15.6, donated by Dr. G. Trinchieri, Wistar Institute of Anatomy and Biology, Philadelphia, PA) at 0 and 1 d before priming with α-GalCer. As a control, the same amount of rat IgG1 ( PharMingen ) was injected intraperitoneally into control mice before injection of α-GalCer. To provide direct evidence that NKT cells are the only target cells for activation by α-GalCer, various lymphoid subsets were isolated from mouse spleen cell suspensions by flow cytometry and cocultured with DCs in the presence of α-GalCer. After 36 h of culture, the supernatants were harvested and their IL-4 and IFN-γ contents were measured by ELISA. Fig. 1 shows that purified NK1.1 + T cells produce higher levels of IL-4 and IFN-γ than unfractionated spleen cells. The IFN-γ produced in these cultures was not derived from classical NK cells, because enrichment of NK1.1 + TCR-α/β − NK cells showed no significant cytokine production. In contrast, NK1.1 + TCR-α/β + cells, which represent the NKT cell population, revealed markedly high levels of IL-4 and IFN-γ production. Although CD4 + T cells produced higher levels of cytokines compared with unfractionated spleen cells, this appeared to be due to the presence of CD4 + NK1.1 + NKT cells, because CD4 + NK1.1 − cells produced neither IL-4 nor IFN-γ in response to α-GalCer. Culture of NK1.1 + TCR-α/β + NKT cells alone or with DCs in the absence of α-GalCer caused no significant production of IFN-γ or IL-4, indicating that DCs are essential for the stimulation of cytokine production by NKT cells. Fig. 2 A shows that coculture of DCs and NKT cells in the presence of α-GalCer results in high levels of IFN-γ production. However, addition of anti–IL-12 mAb into these cultures caused a marked inhibition of IFN-γ production. Such inhibition was not observed when control anti-CD8 rat IgG mAb was added. Therefore, these results indicated that endogenously produced IL-12 by DCs was essential for the early activation of NKT cells by α-GalCer. The effect of mAbs against CD40 and CD40L on the activation of NKT cells by α-GalCer was also investigated . Both anti-CD40 mAb and anti-CD40L mAb greatly inhibited the production of IFN-γ by NKT cells in response to α-GalCer. These findings suggested that direct contact between DCs and NKT cells through CD40/CD40L interactions is critically important for the activation of NKT cells by α-GalCer. To study the requirements for IL-12 production by DCs in these cultures in further detail, IL-12 p70 activity in culture supernatants was measured by ELISA. As shown in Fig. 2 C, DCs produced IL-12 p70 when cultured with NKT cells and α-GalCer. However, DCs did not produce IL-12 p70 when cultured with α-GalCer alone or when cultured with α-GalCer and NK (NK1.1 + TCR-α/β − ) cells. The effect of α-GalCer on the induction of IL-12R mRNA expression in spleen cells was examined by RT-PCR. As shown in Fig. 3 , intravenous injection of α-GalCer into C57BL/6 mice caused the induction of mRNA for both IL-12Rβ1 and IL-12Rβ2 in spleen cells within 4 h. This upregulation of IL-12R was strongly blocked by administration of anti–IL-12 mAb or anti–IFN-γ mAb before injection of α-GalCer . Moreover, the IL-12R induction by α-GalCer was almost completely abolished in both CD1d −/− and Vα14 NKT cell–deficient mice . Thus, these results suggested that CD1d-dependent α-GalCer–induced IFN-γ production by NKT cells may be critically important for the upregulation of IL-12R on NKT cells. To provide direct evidence for this hypothesis, we measured the expression of IL-12R on purified NKT cells that were previously activated in the presence of DCs and α-GalCer, either in vitro or in vivo. Fig. 5 C shows that in vitro activation of spleen cells by DCs plus α-GalCer strongly induced the expression of IL-12R on NKT cells. Similar findings were made when mice were injected in vivo with α-GalCer . C57BL/6 mice were injected intravenously with α-GalCer, and their splenic natural killing activity against YAC-1 cells was determined 24 h later. As shown in Fig. 6 A, a suboptimal dose of neither α-GalCer nor IL-12 was able to activate natural killing activity in vivo. However, combined administration of α-GalCer and IL-12 at a suboptimal dose caused a marked augmentation of natural killing. A similar synergistic effect of α-GalCer and IL-12 was demonstrated for the elevation of serum IFN-γ production. As shown in Fig. 6 B, the administration of α-GalCer plus IL-12 resulted in a strong enhancement of serum IFN-γ levels in C57BL/6 mice compared with mice treated with α-GalCer or IL-12 only. The finding that NKT cells recognize α-GalCer presented by DCs in a CD1d-dependent manner represents a novel recognition mechanism in the immune system ( 15 ). NKT cells, which can produce both IFN-γ and IL-4 ( 16 , 17 ), play an important role in immunoregulation and have been considered to play a central role as innate effector cells involved in both the protection and the onset of immune diseases ( 18 ). The NKT cell ligand α-GalCer has a strong immunopotentiating effect in vivo, and this chemical mediates strong antitumor activity ( 3 – 5 , 9 ). Therefore, it is important to dissect the mechanism by which α-GalCer activates NKT cells. The previous finding ( 3 ) that NKT-deficient mice did not respond to α-GalCer strongly suggested that NKT cells may be the primary target cells to α-GalCer. However, it still remained unclear whether only NKT cells responded to α-GalCer. To answer this question, we used highly purified splenic NK cells, NKT cells, CD4 + T cells, and CD8 + T cells and determined their responsiveness to α-GalCer in the presence of DCs. The data illustrated in Fig. 1 clearly demonstrate that NKT cells are the only cells that respond to α-GalCer ( 3 ). It is surprising that neither classical NK cells nor mainstream CD4 + T cells or CD8 + T cells revealed a significant response to α-GalCer even in the presence of DCs. Together with previous findings ( 3 ), the present data indicate that α-GalCer selectively stimulates NKT cells in the presence of DCs. Recently, the mechanisms of activation of naive CD4 + T cells through interaction with DCs have been examined ( 12 , 19 – 22 ). Cell–cell adhesion between CD4 + T cells and DCs through CD40/CD40L and B7.1/CD28 resulted in the activation of both DCs and T cells, which triggered the production of IL-12 by DCs and IFN-γ by Th1 cells ( 12 , 19 , 20 , 23 – 25 ). Such conditioned DCs were able to prime cytotoxic T cells ( 22 , 26 , 27 ). This recognition system has resemblance to that discussed here. As shown in Fig. 2 , IL-12 production by DCs appears to be essential for NKT cell activation by α-GalCer, because neutralization of endogenously produced IL-12 by anti–IL-12 mAb caused a strong inhibition of IFN-γ production by NKT cells. The important role of CD40/CD40L for the production of IFN-γ in the cocultures of DCs and NKT cells with α-GalCer is also apparent from these experiments . As demonstrated in Fig. 2 C, DCs produce IL-12 only when they are cultured with α-GalCer in the presence of NKT cells, indicating that direct contact between α-GalCer–bound DCs and NKT cells may be essential for IL-12 production by DCs. This interaction may be required for the production of IFN-γ by IL-12–activated NKT cells, because mAbs directed against CD40/CD40L greatly inhibited IFN-γ production by NKT cells . These findings indicate that the interaction of NKT cells with DCs may be very similar to the interaction of helper T cells with DCs ( 22 , 26 , 27 ). Since the interactions between DCs and NKT cells occur very quickly after administration of α-GalCer, NKT cells may be able to condition DCs very early in an immune response, and affect subsequent adaptive responses. In this paper, we also demonstrate that α-GalCer upregulates IL-12R expression in vivo . IL-12R upregulation is blocked by mAbs against IL-12 or IFN-γ and is absent in CD1d −/− and NKT-deficient mice . Moreover, activation of NKT cells in vitro and in vivo results in a strong induction of IL-12Rβ1 and IL-12Rβ2 on these cells . Therefore, we speculate that the following series of events is induced upon culture of α-GalCer with DCs and NKT cells: ( a ) α-GalCer first binds to CD1d molecules on DCs; ( b ) NKT cells recognize α-GalCer–bound DCs via their TCRs and also interact with DCs via CD40/CD40L; ( c ) during this interaction, DCs produce IL-12; ( d ) the endogenously produced IL-12 stimulates IFN-γ production by NKT cells; and ( e ) IFN-γ produced by NKT cells upregulates IL-12R on NKT cells in an autocrine manner. The dramatic synergistic effect of suboptimal α-GalCer and exogenously administered IL-12 indicates that expression of IL-12Rβ1 and β2, detected by quantitative RT-PCR, is functionally upregulated in vivo. Moreover, since this synergistic effect of α-GalCer and IL-12 was not demonstrated in NKT-deficient mice, we conclude that in wild-type mice coadministration of α-GalCer and IL-12 leads to upregulation of IL-12R on CD1-dependent NKT cells. Both α-GalCer and IL-12 have been demonstrated to exhibit potent antitumor activity in vivo. IL-12 has multiple effects on the immune system that are beneficial for the induction of antitumor immunity in vivo ( 28 – 30 ). However, the unexpected severe side effects of IL-12 have made it difficult to use this cytokine in clinical trials ( 31 ). We demonstrated that α-GalCer synergistically acts with small doses of IL-12 in vivo to activate NKT cells and to induce IFN-γ production . These findings suggest that coadministration of α-GalCer with IL-12 could be used as a new approach for tumor immunotherapy. Recent studies have demonstrated that Th1 immunity regulated by IL-12 and IFN-γ plays a critical role in the induction of protective immunity against tumors and infectious agents ( 32 , 33 ). Although NKT cells are involved in both Th1 and Th2 immunity through IFN-γ or IL-4 production, the immunomodulating protocol using α-GalCer and IL-12 preferentially induces NKT cells that produce large amounts of IFN-γ ( 34 ). These NKT cells may facilitate the development of Th1-dominant cellular immunity essential for the induction of protective immunity against tumors and some infectious agents. Recently, it was demonstrated that α-GalCer can stimulate human NKT cells in a CD1d-dependent manner ( 35 , 36 ), indicating that our proposed immunotherapy protocol using α-GalCer and IL-12 will be useful for the application to human immune diseases, including cancer. | Study | biomedical | en | 0.999995 |
10190904 | The mouse IRAK gene in embryonic stem (ES) cells was disrupted by homologous recombination as described in our previous report ( 25 ). In brief, the mouse IRAK gene was disrupted by replacement of a 940-bp region covering exons 5–7 of the gene with a neomycin resistance gene. Chimeric mice were generated from embryos injected with ES cells. Germline mice were obtained from breeding of chimeric male mice with C57BL/6J females. Because the IRAK gene is on X chromosome and the ES cell line was derived from a male embryo, all the germline female mice were heterozygous for the disrupted IRAK gene. IRAK-deficient male mice carrying only the disrupted IRAK gene were obtained from breeding of heterozygous female mice with wild-type littermates. IRAK-deficient female mice were obtained from breeding of heterozygous females with IRAK-deficient males. Thymocytes and splenocytes were stained with CD4- and CD8-specific antibodies ( PharMingen ). Enriched CD4 + T cells before and after 5 d of differentiation were stained with antibodies specific for CD25 and CD44 ( PharMingen ). Cells after antibody staining were analyzed using a FACScan™ ( Becton Dickinson ). CD4 + T cells were purified from lymph node and spleen cells by depletion of B cells and CD8 + T cells using guinea pig and rabbit complements and a combination of antibodies from hybridoma lines J11d, 28-16-8s, and 3-168. Purity of CD4 + T cells in different preparations was ∼90%. Enriched CD4 + T cells were activated with immobilized anti-CD3 ( PharMingen ), which was coated overnight onto 6-well plates at 5 μg/ml. Differentiation of T cells towards Th1 cells was triggered by addition of 5 ng/ml IL-12 (R&D Systems) and 5 μg/ml anti–IL-4 ( PharMingen ) in RPMI medium with 10% FCS. Th2 cell differentiation was driven by supplementing culture medium with 5 ng/ml IL-4 (R&D Systems) and 5 μg/ml of anti–IFN-γ plus anti–IL-12 ( PharMingen ). The cytokine profile of Th1 or Th2 cells was determined by plating the cells at 2 × 10 5 cell/well in 96-well plates that were precoated overnight with 5 μg/ml anti-CD3. Culture supernatants were collected 24 h later for cytokine detection by ELISA. After 5 d of differentiation, Th1 cells were washed once with serum-free RPMI medium and starved for 3 h before stimulation. Cells were treated with different stimuli at 37°C. The different stimuli used in the assays include: IL-18 ( PeproTech, Inc. ), IL-12 (R&D Systems); IL-1β (R&D Systems); TNF-α ( Genzyme Corp. ); PMA and ionomycin ( Sigma Chemical Co. ). After stimulation, cells were lysed for 20 min at 4°C with lysis buffer (100 μl/5 × 10 6 cells) containing 10 mM Hepes, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM Na 3 VO 4 , 10% glycerol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM PMSF, and 1 mM EDTA. Cell lysates for kinase assays and Western blots were collected after centrifugation of samples at 14,000 rpm for 10 min at 4°C. Cell lysates (5 × 10 6 cells in 100 μl of lysis buffer) were immunoprecipitated with JNK1 kinase antibody ( Santa Cruz Biotechnology ). An in vitro kinase assay was performed using glutathione S -transferase (GST)– c -Jun ( Santa Cruz Biotechnology ) as a substrate in a reaction buffer containing 25 mM Hepes, pH 7.4, 25 μM ATP, 10 mM MgCl 2 , 1 μg GST– c -Jun, and 10 μCi [γ- 32 P]ATP for 30 min at 37°C. Samples after kinase assays were separated on 10% SDS-PAGE, transferred to nitrocellulose filters (MSI), and then subjected to autoradiography. Radioactivity of the phosphorylated c -Jun bands were quantitated by PhosphorImager (Molecular Dynamics Inc.). JNK1 protein on blotted filters was detected with a JNK1-specific antibody ( Santa Cruz Biotechnology ). Western blot analyses were carried out as previously described ( 26 ). For detection of IκB-α protein levels, cell lysates (10 5 cells/sample) were separated on 10% SDS-PAGE (Novex) and transferred to nitrocellulose filters. Filters were immunoblotted with an IκB-α–specific rabbit antibody ( Santa Cruz Biotechnology ) and detected with horseradish peroxidase–conjugated rabbit IgG and enhanced chemiluminescence ( Amersham Pharmacia Biotech ). Filters were then stripped by soaking in 0.1 M glycine/HCl, pH 2.6, for 30 min and reprobed with an ERK-2– specific antibody ( Santa Cruz Biotechnology ) for normalization. Cells after treatment with different stimuli were pelleted and taken up in 500 μl ice-cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 100 μM EDTA, 100 μM EGTA, 1 mM dithiothreitol, 500 μM PMSF) and kept on ice for 15 min. Samples were then added with 30 μl of 10% NP-40 followed by centrifugation at 14,000 rpm for 30 s. Nuclear pellets were washed once with buffer A and lysed in 50 μl buffer B (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF) for 30 min at 4°C. Nuclear extracts were collected after centrifugation of the samples at 14,000 rpm for 10 min. Protein concentration was determined by the BCA method ( Pierce Chemical Co. ). The consensus double-stranded oligonucleotide for NF-κB binding ( Santa Cruz Biotechnology ) was labeled using [γ- 32 P]ATP and T4 polynucleotide kinase. Nuclear binding reactions were carried out for 30 min at room temperature in a 20 μl mixture containing 10 μg nuclear extract and 0.5 ng 32 P-labeled oligonucleotide probe (100,000 cpm) in buffer C (5 mM Hepes, pH 7.9, 5 mM MgCl 2 , 1 mM EDTA, 10% glycerol, and 1 μg/ml poly[dI-dC]). Oligonucleotide–protein complexes were separated on 6% polyacrylamide/ 0.5× TBE gels and detected by autoradiography. Total RNA was extracted from cells or spleens with RNAzol (Tel-Test Inc.). RNA was separated on 1% agarose-formaldehyde gels and transferred to nylon membranes ( Amersham Pharmacia Biotech ). Filters were hybridized to cDNA probes specific for IFN-γ ( Clontech ) and IL-18 (Research Genetics, Inc.). cDNA probes were labeled with α-[ 32 P]dCTP ( Amersham Pharmacia Biotech ) by random priming ( Boehringer Mannheim ). Radioactive signals were detected by autoradiography. Filters were then stripped by boiling in 0.1% SDS and rehybridized with glycerol-3-phosphate dehydrogenase cDNA probe ( Clontech ) for normalization. IFN-γ and IL-4 in serum samples and culture supernatants were determined by using commercially available ELISA kits ( Genzyme ) and recombinant cytokines were used as standards provided by the manufacturer. Th1 cells were plated in 96-well plates at 10 5 /well and treated with different cytokines for 24 h. [ 3 H]thymidine (1 μCi/well) was then added for 16 h, and radioactivity incorporated in dividing cells was measured using a Topcount Microplate Scintillation Counter (Packard). Mice were injected intraperitoneally with PBS as controls or PBS with 2 mg of heat-killed P. acnes (Van Kempen Group, Inc.). 7 d later, control mice were injected intravenously with PBS, whereas P. acnes –primed mice were injected with 1 μg LPS ( Sigma Chemical Co. ). Mice were bled 6 h after LPS challenge, and serum IFN-γ levels were measured by ELISA ( Genzyme ). Total RNA was extracted from spleen samples for Northern blot analysis. Mice were injected intraperitoneally daily with PBS alone as controls or PBS containing 1 μg IL-18 or 200 μg poly(I):poly(C) ( Sigma Chemical Co. ) for 2 d. Spleen cells prepared from these mice were incubated with 51 Cr-labeled YAC-1 target cells for 4 h at 37°C at different E/T ratios. After 4 h of incubation, 51 Cr released from target cells was counted using a gamma counter (Packard). Specific lysis was calculated as: (measured 51 Cr release − spontaneous 51 Cr release)/(maximum 51 Cr release − spontaneous 51 Cr release) × 100. Maximum release was obtained by counting acid-lysed target cells. Spontaneous release was obtained by incubating target cells in the absence of effector cells. The Smith strain of MCMV was obtained from the American Type Culture Collection . MCMV stocks were prepared in NIH 3T3 murine fibroblasts, and determination of viral titers was carried out by a standard plaque assay ( 27 ). Mice were intraperitoneally injected with 10 8 PFU of tissue culture–propagated MCMV in 400 μl DMEM. Animals were killed on day 3 after inoculation. Blood sera for cytokine detection were collected by bleeding mice daily starting from day 0 before viral inoculation. Spleens were collected after 71 h of infection for NK cytotoxic assays and RNA extraction. IL-18 by itself has no effect on Th1 differentiation but can synergize IL-12–driven Th1 development ( 4 ). In differentiated helper T cells, IL-18 exerts its biological effects only on Th1 cells but not on Th2 cells ( 4 ). Since IRAK is involved in IL-18 signaling ( 4 , 22 ), we examined helper T cell development and phenotype in IRAK-deficient mice. Development of T cells in the thymus and distribution of mature CD4 + and CD8 + T cells in the primary and secondary lymphoid organs of IRAK-deficient mice appeared to be normal . The vast majority of CD4 + T cells were characterized as CD25 lo CD44 lo naive phenotype and were comparable to the cells harvested from control wild-type mice . Th1 and Th2 cells were prepared by in vitro differentiation of CD4 + T cells from IRAK-deficient and wild-type mice. CD4 + T cells were enriched from lymph node cells and splenocytes by antibody and complement depletion as described in Material and Methods. Enriched CD4 + T cells were activated with immobilized anti-CD3 and differentiation towards Th1 cells was triggered by coculture with IL-12 and anti–IL-4. Th2 cell differentiation was driven by IL-4 and anti–IFN-γ plus anti–IL-12. Activation of IRAK-deficient CD4 + T cells appeared to be normal, as indicated by the upregulation of the activation marker CD25 and the memory T cell marker CD44 . Numbers of blasting T cells after 5 d of differentiation were similar between IRAK-deficient and wild-type T cells (data not shown), suggesting normal proliferation of activated IRAK-deficient T cells. Cytokines expressed by T cells after 5 d of differentiation were characterized by ELISA. Similar to wild-type Th1 cells, IRAK-deficient Th1 cells secreted predominantly IFN-γ, whereas IL-4 was undetectable . IRAK-deficient Th2 cells also showed a typical Th2 cytokine profile similar to that of wild-type Th2 cells, with high amounts of IL-4 but minimal levels of IFN-γ . Our data suggest that IRAK-deficient T cells can differentiate into Th1 or Th2 effector cells. The stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases JNK and p38 are rapidly activated by proinflammatory cytokines such as IL-1 and TNF-α ( 24 , 25 , 28 ). IL-18 stimulation of Th1 cells also results in JNK activation ( 2 ). JNK plays a role in induction of activator protein (AP)-1–dependent genes via phosphorylation of the transcription factor c -Jun ( 28 , 29 ). We have shown previously that IRAK-deficient fibroblasts are defective in IL-1–mediated JNK activation ( 25 ). To determine the role of IRAK in IL-18–induced JNK activation, JNK activity in Th1 cells was studied by immunoprecipitating JNK1 with a specific antibody followed by an in vitro kinase assay using the GST– c -Jun protein as a substrate. Activation of JNK was minimal in IL-18–treated IRAK-deficient Th1 cells as opposed to the significant JNK activation observed in wild-type cells . In contrast, TNF-α induced similar levels of JNK activity in both wild-type and IRAK-deficient Th1 cells, whereas IL-1β did not have any effects on either samples . IL-18 and IL-1 have been suggested to act on Th1 and Th2 cells, respectively, to induce signaling and cellular responses ( 4 ). In our studies, Th1 cells do not respond to IL-1β in JNK activation and this is consistent with a previous observation ( 4 ), which suggested that IL-1 does not induce signaling in Th1 cells due to the lack of IL-1 receptor expression. Our results indicate that the defect in JNK activation observed in IRAK-deficient Th1 cells is IL-18 specific. IL-18 binding to its receptor mediates activation of NF-κB ( 4 , 22 ). In unstimulated cells, NF-κB is present in the cytoplasm as an inactive complex sequestered by its inhibitory partners, IκB ( 30 ). Upon activation, IκB proteins are phosphorylated by IκB kinases IKK1 and IKK2, and subsequently degraded by proteosomes to allow nuclear translocation and activation of NF-κB ( 30 – 32 ). To determine the role of IRAK in IL-18–induced NF-κB activation, IκB-α protein levels were determined in Th1 cells stimulated with IL-18 for different time courses . Reduced levels of IκB-α due to protein degradation were observed in both wild-type and IRAK-deficient cells after IL-18 treatment . Maximum degradation of IκB protein occurred 15 min after stimulation and returned to 80% of original levels within 60 min of stimulation. The extent of IκB-α protein degradation mediated by IL-18 appeared to be less in IRAK-deficient Th1 cells as compared with that in wild-type cells. Similar results were observed when wild-type and IRAK-deficient Th1 cells were stimulated with different concentrations of IL-18 (data not shown). This result was further confirmed by NF-κB DNA binding activity in nuclear extracts determined by mobility shift assay. IL-18–induced NF-κB activation in both IRAK-deficient and wild-type cells, but NF-κB DNA binding activity was slightly lower in IRAK-deficient cells than in wild-type cells . Stimulation with TNF-α or phorbol ester plus ionomycin induced comparable levels of NF-κB activation in both cell types, indicating that the partial defect in NF-κB activation observed in IRAK-deficient cells is restricted to IL-18 stimulation. These results suggest that involvement of IRAK in IL-18–mediated NF-κB activation is dispensable. IL-18 induces IFN-γ production from Th1 cells ( 1 , 5 ). Stimulation of Th1 cells with a combination of IL-18 and IL-12 results in synergistic induction of IFN-γ production ( 4 ). To determine whether IRAK is required for induction of IFN-γ by IL-18 itself or in combination with IL-12, IFN-γ mRNA expression was determined by Northern blot analysis. IFN-γ was significantly induced by IL-18 in wild-type cells but its induction in IRAK-deficient cells was minimal . A suboptimal dose of IL-12 was used in our experiments, which induced minimal amounts of IFN-γ, and no difference was observed between IRAK-deficient and wild-type cells. Synergistic induction of IFN-γ expression by a combination of IL-18 and IL-12 was substantially decreased in IRAK-deficient cells as compared with wild-type cells. This result shows that IRAK is required for optimal induction of IFN-γ by IL-18. It has been demonstrated that treatment with LPS in P. acnes –sensitized mice results in a significant increase in serum IFN-γ ( 33 ). IL-18–deficient mice exhibited a minimal increase in serum IFN-γ under these conditions ( 3 ). To determine the role of IRAK in IL-18–dependent IFN-γ production in vivo, IRAK-deficient mice were tested in this experimental system. Mice were injected intraperitoneally with heat-killed P. acnes and 7 d later were injected intravenously with LPS. IFN-γ in the serum was detected by ELISA 6 h after LPS treatment. Serum IFN-γ levels were significantly lower in IRAK-deficient mice as compared with wild-type animals . Consistent with these data, IFN-γ mRNA expression in the spleen was substantially reduced in IRAK-deficient mice. In contrast, induction of IL-18 mRNA expression was comparable between wild-type and IRAK-deficient animals . These results suggest that the reduced IFN-γ production in IRAK-deficient mice is not due to a change in IL-18 levels but rather originated from defects in IL-18 signaling. Similar to induction in IFN-γ production, proliferation of Th1 cells was also enhanced by IL-18 or IL-12, and synergized by the combination of both ( 4 ). The effect of IL-18 and its synergism with IL-12 on proliferation of IRAK-deficient Th1 cells was studied. Wild-type and IRAK-deficient Th1 cells were treated with different concentrations of IL-18, IL-12, or IL-18 plus IL-12. Proliferation of Th1 cells after 24 h of stimulation was determined by [ 3 H]thymidine uptake. As shown in Fig. 5 , proliferation of wild-type Th1 cells was significantly enhanced by IL-18 in a dose-dependent manner, whereas the effect of IL-18 on proliferation of IRAK-deficient cells was minimal. At concentrations as low as 2 ng/ml, IL-12 stimulated proliferation of both wild-type and IRAK-deficient cells to maximal extent. Combination of IL-18 and IL-12 resulted in a synergistic proliferative response in both cell types, and no significant difference was observed. Consistent with the defects shown in IFN-γ expression, IRAK-deficient cells are also impaired in proliferative response to IL-18, confirming the role of IRAK in IL-18–mediated cellular responses. IL-18 has been shown to enhance NK cell cytotoxicity ( 1 ). Reduced NK activity was reported in IL-18– deficient mice ( 3 ). To determine whether IRAK is involved in IL-18–induced NK activity, mice were injected intraperitoneally with IL-18 for 2 d consecutively, and NK activity in splenocytes was assayed using 51 Cr-labeled YAC-1 as target cells. Basal NK cell activities in PBS injected wild-type and IRAK-deficient mice were comparable . IL-18 injection resulted in significant increase in NK activity in wild-type animals but its effect in IRAK-deficient mice was minimal . However, injection of the double-stranded RNA poly(I):poly(C), an inducer of IFNs, resulted in a pronounced NK activity in both IRAK-deficient and wild-type animals , indicating that IRAK is required specifically for IL-18–mediated induction of NK cytotoxic activity. NK cells are the major effector cells in the early defense against viral infections. Both NK cytotoxicity and IFN-γ production by NK cells are induced in mice upon MCMV infection ( 34 ). IL-12 has been shown to be responsible for induction of IFN-γ in NK cells ( 35 ), whereas possible roles of IL-18 in NK activities during MCMV infection have not been reported. IRAK-deficient mice were infected with MCMV to study the involvement of IRAK in NK activity during viral infection. Splenocytes obtained from mice on day 3 of MCMV infection were assayed for NK cytolytic activity using 51 Cr- labeled YAC-1 cells as targets. Dramatic increase in NK cytotoxicity was observed in both MCMV-infected wild-type and IRAK-deficient mice and no significant difference was found between the two types of mice . IFN-γ produced by NK cells during MCMV infection can be detected as an increase in serum IFN-γ levels. Kinetics of IFN-γ increase in sera during the first 3 d of MCMV infections was studied. In both wild-type and IRAK-deficient mice, IFN-γ levels peaked at 45 h of infection . However, maximal IFN-γ levels in IRAK-deficient mice were significantly lower than those in wild-type control mice . Similar results were obtained in studies of IFN-γ mRNA expression. IFN-γ mRNA in the spleens was undetectable in uninfected mice but was induced significantly in MCMV infected mice . IFN-γ mRNA induced by MCMV was less significant in IRAK-deficient mice than that in wild-type mice. IL-18 expression in response to MCMV infection was also studied. IL-18 mRNA was expressed at low levels in the spleens of uninfected mice and was induced strongly on day 3 of MCMV infection . IL-18 expression in IRAK-deficient mice was comparable to that in wild-type mice under healthy and viral-infected conditions . Thus, impairment in viral-induced IFN-γ production in IRAK-deficient mice is not due to the levels of IL-18 expression, suggesting that other mechanisms, such as IL-18 signaling, may be responsible for the defects. We have demonstrated IL-18 signaling defects in IRAK-deficient Th1 cells. Impairment is significant in JNK activation but much less obvious in NF-κB induction. The partial activation of NF-κB suggests that other mechanisms can compensate for the function of IRAK. However, the role of IRAK in JNK activation is essential. We have previously reported that IRAK-deficient fibroblasts are defective in both NF-κB and JNK/p38 pathways induced by IL-1 ( 25 ). The defects in IL-18 signaling that we observed here are similar to the defects in IL-1 signaling ( 25 ). Impairment in NF-κB activation can be overcome by high concentrations of IL-1, but similar treatment cannot correct defects in JNK activation ( 25 ). Taken together, our results suggest that IRAK is used similarly by both IL-18 and IL-1 in mediating intracellular signaling. IL-1 signaling leading to NF-κB activation has been relatively well characterized. Upon IL-1 binding to its receptor, IRAK is rapidly recruited to the receptor complex via MyD88 ( 20 ). Activated IRAK interacts with TNF receptor–associated factor (TRAF)6, which in turn activates NF-κB–inducing kinase (NIK) ( 36 , 37 ). NIK is involved in the NF-κB pathway by activating IκB kinases (IKKs) ( 31 , 32 , 38 , 39 ). Activated IKKs phosphorylate IκB for degradation, allowing nuclear translocation of NF-κB for gene induction ( 30 ). However, details of IL-1–mediated signaling leading to JNK activation are still unclear. It has been reported that only MyD88 but not TRAF6 is essential for JNK activation ( 40 ). Since IRAK is positioned between MyD88 and TRAF6 in the signaling cascade, IRAK could be the bifurcating molecule for both NF-κB and JNK pathways. IL-18 stimulation also induces the stress-activated MAP kinase JNK ( 2 ). The defects in JNK activation in IRAK-deficient cells indicate that IRAK is essential for JNK activation but intermediary molecules linking IRAK to JNK have not been identified. Recent reports on IL-1 signaling also suggest additional complexity and divergence in this pathway. In addition to IRAK, other proteins, including IRAK homologue IRAK2, are reported to interact with the IL-1 receptor ( 19 , 41 ). IRAK2 also interacts with MyD88 and TRAF6 ( 19 ). A dominant negative form of IRAK2 mutant blocks MyD88-induced NF-κB activation ( 19 ). Although overexpression of IRAK in transfection studies has been reported to activate NF-κB but not JNK ( 42 ), our studies demonstrate that IRAK is essential for IL-18–mediated activation of JNK but its role in NF-κB pathway is less critical. In the absence of IRAK, NF-κB activation can still occur, possibly mediated by other related kinases such as IRAK2. The relative role of IRAK and IRAK2 in inducing JNK and NF-κB downstream of MyD88 is still unclear and the involvement of IRAK2 in IL-18 signaling remains to be elucidated. IL-18 alone does not support Th1 differentiation, but it can potentiate Th1 development driven by IL-12 ( 4 ). In differentiated Th1 cells, IFN-γ production can be induced by IL-18 or IL-12 alone, and this effect can be further synergized by a combination of both cytokines ( 4 ). We observed no defect in Th1 differentiation of IRAK-deficient cells mediated by IL-12, confirming that IL-18 signaling does not play an essential role in Th1 development. IL-18–induced IFN-γ expression in IRAK-deficient Th1 cells is significantly decreased, although NF-κB activation is only partially impaired. Increase in serum IFN-γ levels as a result of a Th1 response to P. acnes and LPS treatment, or an NK response in the early phase of MCMV infection, was also reduced significantly in IRAK-deficient mice. The results suggest that optimal gene induction by IL-18 requires proper activation of multiple signaling pathways including NF-κB and JNK. Dramatic decrease in IFN-γ production in IRAK-deficient cells may have resulted from the impairment in JNK activation even though NF-κB activity was not severely affected. We also observed similar phenomena in our previous studies; IL-6 induction in IRAK-deficient fibroblasts in response to IL-1 treatment was significantly reduced due to defects in JNK/p38 activation despite minimal impacts on NF-κB activation ( 25 ). The involvement of both JNK and NF-κB pathways may imply that optimal gene expression depends on the binding of multiple transcription factors including NF-κB and AP-1 onto the promoter regions of the target genes. It is also possible that the transactivation potential of NF-κB is modulated by the kinase activity of JNK and the other MAP-related kinase p38, as suggested in studies of TNF-induced IL-6 gene expression ( 43 ). IL-18 and IL-12 share many biological properties, although different signaling pathways are used by the two cytokines. Transcription factor STAT4 is activated by IL-12 whereas NF-κB and AP-1 are activated by IL-18. The binding sites of these transcription factors within the IFN-γ promoter region have been identified ( 44 ). IL-18 alone can directly induce IFN-γ promoter activity via AP-1, whereas IL-12– mediated induction of the promoter activity requires both AP-1 and STAT4 ( 44 ). Synergistic expression of IFN-γ by IL-18 and IL-12 probably results from the interplay of multiple transcription factors in differential regulation of IFN-γ promoter activity. A strong synergistic effect in IFN-γ induction and cell proliferation was observed in IRAK-deficient Th1 cells when treated with combination of IL-12 and IL-18. The results suggest that minimal activation of NF-κB and JNK by IL-18 in IRAK-deficient cells is sufficient to function synergistically with IL-12 to achieve a significant synergistic response. MCMV infection in mice results in a strong NK response in the early phase of infection before the onset of T and B cell responses ( 34 ). Induction of NK cell IFN-γ production and cytotoxicity peaks on day 2–5 of infection ( 34 , 45 ). IFN-γ production by NK cells is the major defense mechanism in controlling MCMV replication ( 34 ). It has been well characterized that in MCMV infection IL-12 plays a major role in NK cell IFN-γ production, whereas induction of NK cytotoxicity is regulated by IFN-α/β ( 35 , 46 ). Although IL-18 is known to be involved in NK cell function, the role of IL-18 in NK responses during MCMV infection has not been investigated. We have undertaken this study to understand the importance of IRAK in IL-18–mediated responses in a viral infection. MCMV-infected IRAK-deficient mice have a normal induction of IL-18 expression but exhibited a significant decrease in IFN-γ induction and in serum IFN-γ. The results in this study suggest that IL-18 plays a distinct role in IFN-γ production during the NK response to MCMV infection and that its function cannot be overcome completely by IL-12 or other mechanisms. However, we cannot rule out the possibility that IRAK may also play a role in viral infections via its involvement in other related receptors in addition to the IL-18 receptor. Although we have demonstrated a defect in IL-18–induced NK cytotoxicity in IRAK-deficient mice, MCMV infection or poly(I): poly(C) injection triggers a normal NK cytotoxicity in these mice. Our results suggest that the role of IL-18 in NK cytotoxicity can be compensated by IFN-α/β or other mechanisms during MCMV infection. Studies are underway in our laboratory to understand the mechanisms of MCMV-induced immune responses in IRAK-deficient mice. | Study | biomedical | en | 0.999996 |
10190905 | Samples of human cord blood (CB) were obtained from placental and umbilical tissues and diluted (1:3) in IMDM ( GIBCO BRL ). The mononuclear cells were collected by centrifugation on Ficoll-paque ( Amersham Pharmacia Biotech ). CD34 + CD38 − Lin − cells were collected using our standard protocol ( 22 , 23 ). CB cells were first enriched for CD34 + cells by negative selection using a cocktail of lineage (Lin) antibodies and the StemSep device as described by the manufacturer (Stem Cell Technologies, Inc.). These cell fractions were then stained with anti–human CD34-FITC and anti–human CD38–PE ( Becton Dickinson Immunocytometry Systems ), analyzed, and sorted on a FACStar Plus ™ ( Becton Dickinson ). The sorting gates used were similar to those shown previously ( 34 , 38 ). Data acquisition and analysis were performed using CELLQuest™ software ( Becton Dickinson ). Purified cells were collected after sorting in 500-μl tubes, and mRNA was extracted from 1,000 cells for each PCR reaction using a purification kit ( Amersham Pharmacia Biotech ). The mRNA was reverse transcribed into cDNA by standard methods using Superscript II ( GIBCO BRL ) as the reverse transcriptional enzyme. PCR was performed for the detection of transcripts using a Perkin-Elmer 9700 cycler with the indicated specific primers for 40 cycles. Primer sequences used for transcript detection for SMADs and ALK receptor were as follows: SMAD-1F, 5′-CGAATGCCTTAGTGACAG-3′, and SMAD-1R, 5′-GAGGTGAACCCATTTGAG-3′; SMAD-4F, 5′-AGGTGAAGGTGATGTTTG-3′, and SMAD-4R, 5′-GCTATTCCACCTACTGAT-3′; SMAD-5F, 5′-TGTTGGTGGAGAGGTGTA-3′, and SMAD-5R, 5′-AGATATGGGGTTCAGAGG-3′; ALK-3F, 5′-ACCATCGGAGGAGAAACT-3′, and ALK-3R, 5′-CTGCTGCGCTCATTTATC-3′; ALK-6F, 5′-AAGTTACGCCCCTCATTC-3′, and ALK-6R, 5′-TGATGTCTTTTGCTCTGC-3′. Human clonogenic progenitors were assayed under standard conditions as shown previously, which included the addition of 10% 5637 conditioned medium as a source of cytokines ( 38 ). In brief, 100–500 purified cells were plated in methylcellulose cultures aliquoted in 1-ml vol in 35-mm suspension culture dishes and incubated at 37°C. After 10–14 d, clonogenic progenitors were scored according to standard criteria ( 38 ). CD34 + CD38 − Lin − cells were incubated in serum-free conditioned medium shown previously to maintain primitive human populations ( 38 ). In brief, conditioned medium is comprised of 50 μl of IMDM supplemented with 1% BSA (Stem Cell Technologies, Inc.), 5 μg/ml of human insulin (Humulin R; Eli Lilly and Co.), 100 μg/ml of human transferrin ( GIBCO BRL ), 10 −4 M β-mercaptoethanol, and GFs. GF cocktail was used at final concentrations of 300 ng/ml of stem cell factor (SCF; Amgen) and Flt-3 ( Immunex ), 50 ng/ml of G-CSF (Amgen), and 10 ng/ml of IL-3 (Amgen) and IL-6 (Amgen). Cells were cultured in flat-bottomed suspension wells of 96-well plates (Nunc), incubated for the appropriate times as indicated, at 37°C and 5% CO 2 , and 50 μl of fresh GF cocktail was added to each well every other day. Mesodermal factors were added to obtain final concentrations as indicated. Individual factors were obtained from the following sources: TGF-β1 and TGF-β1–3 neutralizing antibody (R&D Systems), BMP-2 (gift from Dr. Vicki Rosen, Genetics Institute, Cambridge, MA), BMP-4 (gift from Dr. Steve Neben, Genetics Institute), and BMP-7 (gift from Dr. Kuber Sampath, Creative Biomolecules, Inc., Boston, MA). Cells were transplanted by tail vein injection into sublethally irradiated NOD/LtSz-scid/scid (NOD/SCID) mice (375-cGy 137 Cs) according to our standard protocol ( 36 , 39 ). In all cases, cells were cotransplanted with irradiated nonrepopulating CD34 − Lin + cells as accessory cells ( 34 , 38 ). Mice were killed 8 wk after transplantation, and BM cells were collected from femurs, tibiae, and iliac crests. High molecular weight DNA was isolated from the BM of transplanted mice, and the percentage of human cells was determined by probing with a human chromosome 17–specific α-satellite probe as described previously ( 36 , 39 ). The level of human cell engraftment was quantified by visual inspection of film developed from Southern blot by comparing the characteristic 2.7-kb band with human/mouse DNA mixture controls (limit of detection, 0.05% human DNA) that provided a linear signal response. In some cases, BM of transplanted mice was analyzed by staining with human panleukocyte marker CD45 to detect the presence of human hematopoietic cells using the FACScan ® as described previously ( 34 , 36 , 38 , 39 ). Two- or three-color flow cytometric analysis was performed as shown previously to ensure that human engrafting cells contained multiple lineages (data not shown). The data were analyzed by the unpaired, two-tailed Student's t test assuming a Gaussian distribution (parametric test) using Prism ® software, version 2.0 (GraphPad). TGF- β receptors have previously been shown to be expressed on highly purified primitive hematopoietic populations in which soluble TGF-β is capable of regulating proliferation and progenitor cell content ( 19 , 20 ). Our previous studies demonstrated that highly purified SRCs derived from both human CB and BM were found in the fraction of CD34 + CD38 − Lin − cells ( 34 , 35 , 38 ). The type I BMP receptors ALK-3 and ALK-6 are capable of binding BMPs in the absence of type II receptors, and it has been suggested that these receptors may be capable of ligand selection and may be specific for BMPs ( 15 , 40 ). Reverse transcription (RT)-PCR analysis demonstrated that both ALK-3 and -6 are expressed in primitive CD34 + CD38 − Lin − cells isolated from human CB and BM tissue . Detection of ALK-3 and -6 expression in BM samples was more difficult compared with CB-derived primitive populations at similar RT-PCR conditions, suggesting lower expression in BM versus CB. SMAD-1 and -5 are restricted for BMP signaling, whereas SMAD-4 is a shared mediator of TGF-β signaling, and acts as a common partner with pathway-specific SMADs ( 25 , 26 ). Purified CD34 + CD38 − Lin − cells isolated from human CB, BM, mobilized peripheral blood (M-PB), and human BM-derived stroma express SMAD-1 and -5 transcripts . SMAD-4 expression was found in all sources of primitive cells, but was more easily detected in BM ( n = 2) than in CB samples ( n = 4) and was barely detectable in M-PB ( n = 2). In summary, transducers of the BMP signaling pathway are expressed in primitive subfractions of both embryonic and adult human hematopoietic tissue, suggesting that candidate human stem cell populations have the capability of responding to BMPs. The proliferative response of CD34 + CD38 − Lin − cells in response to BMP treatment was determined by comparing the number of cells obtained after 3 d of culture in the presence or absence of BMPs to the number originally seeded at day 0 . Cells were seeded in ex vivo culture conditions with serum-free media (control SF, which we had previously designed to allow for the expansion of primitive human blood cells ) and were compared with cultures in which TGF-β, BMP-2, -4, -7, or 5% serum was added . The addition of TGF-β inhibited the proliferative response seen in control SF conditions by 80% but did maintain the number of cells initially incubated, whereas TGF-β neutralizing antibody had no additional effect on total cell number compared with control conditions . Consistent with effects on cell growth, treatment with TGF-β maintained the number of progenitors capable of producing CFCs after 3 d of culture, whereas TGF-β antibody had no effect on CFC capacity . BMP-2 and -7 had a modest effect on cell growth at concentrations of 5 ng/ml; however, at higher concentrations (50 ng/ml) both BMP-2 and -7 treatment inhibited cell proliferation similar to TGF-β. The inhibitory growth effect of BMP-2 and -7 is consistent with decreased CFC content of treated cultures. BMP-2 and -7 decreased the CFC capacity of CD34 + CD38 − Lin − cells in a dose-responsive manner; however, at 5 ng/ml BMP-7 was less effective than BMP-2 . In the cases of TGF-β, BMP-2, and BMP-7, changes in total cell number correlated with changes in CFC content but did not selectively alter the specific type of progenitor detected, demonstrating that BMPs do not affect lineage commitment. This suggests that BMP-2 and -7 are capable of modulating proliferation of primitive CD34 + CD38 − Lin − cells in a manner that does not alter the developmental program and differentiation capacity of primitive cell populations. Addition of 5% serum caused a massive proliferative response along with a dramatic decrease in the number of cells capable of producing progenitors, indicative of differentiation induction . Treatment of CD34 + CD38 − Lin − cells with BMP-4 invoked a unique response compared with TGF-β, BMP-2, and BMP-7. The addition of BMP-4 at 5 ng/ml inhibited the cell growth of CD34 + CD38 − Lin − cells, in contrast to the effects observed using BMP-2 and -7 at similar concentrations . Increasing BMP-4 concentrations to 50 ng/ml was toxic to CD34 + CD38 − Lin − cells (data not shown). Concentrations of 25 ng/ml of BMP-4 did not alter cell viability and were capable of inducing an increase in cell number over control SF conditions . The ability of CD34 + CD38 − Lin − cells to produce CFCs in response to BMP-4 was also dose dependent. Although 5 ng/ml of BMP-4 inhibited proliferation of CD34 + CD38 − Lin − cells, CFC content was only slightly decreased compared with control, resulting in increased frequency. CD34 + CD38 − Lin − cells treated with 25 ng/ml of BMP-4 dramatically expanded CFCs compared with control SF conditions . These data indicate that members of the TGF-β family, including those in the BMP subfamily, are capable of modulating proliferation and differentiation of primitive human blood cells and that the effects are specific to the dose and subtype of BMP ligand. To determine whether BMP treatment affected the differentiation program of primitive human CD34 + CD38 − Lin − cells, cultures were analyzed by flow cytometry for changes in CD34 and CD38 expression . Similar to that shown previously ( 38 ), control SF cultures induced modest differentiation of CD34 + CD38 − Lin − cells into CD34 + CD38 + cells , whereas the addition of 5% serum induced a differentiation response as demonstrated by the acquisition of CD38 and loss of CD34 expression. Both TGF-β and TGF-β neutralizing antibody had little effect on the CD34 + CD38 − phenotype and remained similar to control cultures . Members of the BMP subfamily caused changes in CD34 + CD38 − phenotype compared with control SF conditions . At concentrations of 5 ng/ml, both BMP-2 and -7 had a potent differentiation effect on CD34 + CD38 − Lin − cells as demonstrated by the acquisition of CD38; however, the population remained relatively immature since the cells were all CD34 positive. At higher concentrations of 50 ng/ml, both BMP-2 and -7 maintained equivalent numbers of CD34 + CD38 − cells compared with control SF conditions . Phenotypic analysis of CD34 + CD38 − Lin − cells after treatment with BMP-4 was distinct from that observed for BMP-2 and -7. At low doses, BMP-4 induced a complete differentiation of CD34 + CD38 − cells into CD34 + CD38 + cells, whereas at higher doses two populations developed: one that had differentiated and one that maintained a primitive phenotype . To further investigate the unique response of CD34 + CD38 − Lin − cells to BMP-4, phenotypic analysis was extended to 6 d of ex vivo culture . Parallel cultures of CD34 + CD38 − Lin − cells incubated in control SF conditions for 6 d differentiated into CD34 + CD38 + cells, similar to the differentiation response seen when low concentrations of 5 ng/ml of BMP-4 were added . In contrast, the high concentration of BMP-4 treatment resulted in the maintenance of a significant proportion of primitive CD34 + CD38 − cells. These changes in the phenotype of CD34 + CD38 − Lin − cells in response to BMP treatment illustrate that these factors are capable of modulating the developmental program of primitive subsets of CD34 + cells. Consistent with effects on proliferation and CFC capacity, BMP-4 is capable of inducing distinct dose-dependent effects on the differentiation program of human CD34 + CD38 − Lin − cells compared with other members of the TGF-β superfamily tested. Using the SRC assay, we have previously demonstrated that serum-free ex vivo cultures were the only conditions capable of maintaining pluripotent repopulating cells for as long as 4 d before transplantation ( 38 ). However, even under these conditions, no SRCs were present after 9 d although CFCs and CD34 + cells expanded enormously during this time period. These previous results indicated that even these optimized cultures had limited ability to expand or even maintain SRCs. Accordingly, we have assessed the effect of BMP treatment on SRC maintenance and expansion. Wells were seeded initially with 700–1,000 CD34 + CD38 − Lin − cells in control SF conditions representing 1 or 2 SRCs. BMPs were added as indicated, and the entire contents of each well were then transplanted into NOD/SCID mice at 2, 4, and 6 d. Human cell engraftment was determined 8 wk after transplant, and results are summarized in Table I . The frequency of mice engrafted after transplantation of cells cultured for 2 d in ex vivo cultures was similar in all treatment groups, with the exception of cultures containing 5% serum, which were unable to sustain SRCs after 2 and 4 d (Table I ). The addition of serum caused the majority of CD34 + CD38 − cells to acquire CD38 and/or lose CD34 cell surface expression after 4 d . This differentiation shift suggests that the SRCs have most likely undergone differentiation into more mature cells that no longer repopulate. However, since primitive CD34 + CD38 − cells were present in serum-containing cultures, the loss of SRCs is most likely associated with maturation that occurred independent of CD38 acquisition. Similar results have also been obtained in a separate study examining the loss of SRCs during culture for retroviral transduction, and demonstrate that there is dissociation between phenotype and function as a consequence of downregulation of CD38 in differentiated cells after culture in serum (our unpublished data). All mice transplanted with purified cells cultured for 4 d in control SF conditions were repopulated (100%). However, cultures containing ligands at concentrations having inhibitory effects on cell growth and CFC capacity, such as TGF-β, 50 ng/ml of BMP-2 and -7, and 5 ng/ml of BMP-4 , all resulted in a decrease in the frequency of engrafted animals, suggesting a loss of SRCs during culture (Table I ). In contrast, but consistent with mitogenic responses detected by cell growth and CFC capacity, the addition of TGF-β neutralizing antibody, 5 ng/ml of BMP-2 and -7, or 25 ng/ml of BMP-4 allowed for the maintenance of SRCs after 4 d of culture as indicated by 100% engraftment frequency in transplanted animals. Since all mice transplanted with control SF cultures at 4 d of culture were also engrafted, it was difficult to determine whether the addition of BMPs was affecting human repopulating cells. By using similar techniques of limiting dilution analysis employed in our previous studies ( 34 , 38 ), we compared cultures treated with BMPs or TGF-β to assess effects on the number of SRCs at day 4; no significant differences in the frequency of SRCs were found (data not shown). To determine whether BMPs were capable of affecting the survival of SRCs, cultures were extended for up to 6 d. After 6 d of ex vivo culture, SRCs could not be detected under SF conditions. In contrast, one out of four cultures containing TGF-β neutralizing antibody or 5 ng/ml of BMP-2 contained repopulating cells. The most dramatic effect was seen in cultures containing 25 ng/ml of BMP-4, where as few as 700 CD34 + CD38 − Lin − cells cultured under these conditions for 6 d were capable of engrafting 5 out of 6 mice (83%; Table I ). The percentage of human chimerism in the BM of all positive mice shown in Table I ranged between 0.1 and 1%. These results are consistent with our previous studies in which transplantation of one SRC enriched in purified CD34 + CD38 − cells at limiting dose allowed for similar levels of human engraftment ( 34 , 38 ). Further extension of ex vivo cultures containing 25 ng/ml of BMP-4 to 8 d resulted in the loss of SRCs (data not shown). These data demonstrate the novel role of BMPs in regulating the repopulating function of primitive human hematopoietic cells and demonstrate that BMP-4 acts as a survival factor for candidate human stem cells. The development of serum-free ex vivo culture systems together within xenogenic transplant systems that are capable of detecting human repopulating cells has provided a method for the further identification of factors regulating the developmental program of candidate human stem cells (SRCs ). In this report, we provide the first evidence for the role of BMPs in the modulation of the developmental program of primitive subfractions of CD34 + CD38 − Lin − cells that contain SRCs. Treatment with BMP-2, -4, and -7 resulted in dose-dependent effects on the growth, differentiation, and repopulating function of CD34 + CD38 − Lin − cells. Evidence of the regulatory role of BMPs was best demonstrated by BMP-4 treatment. At low concentrations of BMP-4, rapid differentiation was seen as well as loss of SRCs. At high doses, some differentiation occurred, but a significant proportion of primitive CD34 + CD38 − Lin − cells remained and more importantly SRCs were present. BMP-4 was capable of extending the duration of stem cell activity in culture for an additional 2 d in comparison with all other previously optimized conditions using cytokines believed to act on primitive blood cells. Thus, BMP-4 is acting as a survival factor, preserving stem cell function under conditions that normally lead to stem cell loss. The expression of pathway-restricted SMAD-1 and -5, together with expression of BMP receptors ALK-3 and -6, suggest that the mechanism of BMP action is due to the specific activation of the BMP pathway that is distinct from TGF-β signaling. In addition, BMP-2 and -4 normally induce similar cellular responses, and therefore the differential effects of BMP-2 compared with BMP-4 on primitive hematopoietic tissue shown here are unique, and remain to be tested in other species and tissue types using similar in vivo model systems for primary tissue. This unique response of human blood stem cells to BMP-4 ligand may be due to a previously unreported receptor and/or inhibitory molecule mediating BMP-4 signals that is expressed by this population of rare blood cells. Alternatively, the divergent effect of BMP-2 and -4 may not be at the receptor level and may be due to the synergistic effects of BMP-4 with other cytokines used in this ex vivo culture system, which do not have overlapping effects on BMP-2–specific pathways. The differential response of BMP-2 and -4 may not diverge at the level of intercellular signaling in individual cells, but could be due to an intrinsic heterogeneity of BMP receptor expression within the cells that comprise this population. At day 3 and 6 of ex vivo culture, BMP-4 was capable of inducing a differentiation response shown by the acquisition of CD38, but also of maintaining the primitive phenotype of a potentially distinct subset of CD34 + CD38 − cells. These results are most easily interpreted by the existence of two differentially responsive populations that are heterogeneous at the level of BMP binding proteins. Evidence from other gene transfer and cell purification studies has already suggested that CD34 + CD38 − Lin − cells are heterogeneous and that there is a hierarchy of stem cells within this cell fraction ( 35 ). To address this possibility, it would be necessary to develop flow cytometric methods to detect subpopulations within CD34 + CD38 − Lin − fractions using fluorochrome-conjugated BMP ligands or antibodies to BMP receptors. Reagents to perform these experiments are currently being developed. The results reported here, together with studies using other developmental systems, underscore the role of BMP-4 in primitive hematopoietic tissue and demonstrate that BMPs continue to regulate blood development well after tissue specification ( 13 , 41 ). Thus, an entirely new avenue remains to be explored to identify this new biological mechanism of stem cell regulation. Moreover, ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications, including gene therapy, tumor cell purging, and stem/progenitor cell expansion ( 42 ), and therefore the identification of this novel class of stem cell regulatory molecules opens the way to developing these clinical applications. Since current ex vivo culture systems for human blood cells are limited in their ability to maintain stem cells in vitro ( 38 , 42 ), the ability to extend the period in which repopulating cells can be maintained in culture with the addition of factors such as BMP-4 represents a significant advance in these systems. Based on the novel role of this family of molecules in the regulation of primitive hematopoietic tissue, this study establishes the foundation for the use of these and other mesodermal regulators in the manipulation of human stem cells in a clinical setting. | Study | biomedical | en | 0.999998 |
10190906 | 42D1 mAb (rat IgG2a) has been described previously ( 20 ) and 27D6 mAb (rat IgM) was another clone obtained from the same fusion. TC16-28C8 and TC16-40H2 mAbs (both rat IgG1) were produced by immunizing female Lewis rats with a human Fcγ1 fusion protein of ILT6, a receptor that naturally lacks a transmembrane domain. Reactivities of 40H2, 28C8, 42D1, and 27D6 are detailed in the legend to Fig. 5 , based on flow cytometry experiments using the panel of cell surface ILT receptor transfectants available for this study . Baf3 cells transfected with CD94/NKG2A ( 29 ), CD94/ NKG2C/DAP12 ( 29 ), or KIR receptors ( 30 ); Jurkat cells transfected with ILT3 ( 21 ); RBL cells transfected with ILT4 or ILT5 ( 20 ); and P815 cells transfected with ILT1 ( 22 ) have been described previously. Baf3 cells transfected with ILT2 were generated as previously described ( 29 ). RBL cells transfected with ILT8, an activating receptor similar to ILT1, were produced as previously described ( 20 ). Using HLA-G*01012 cDNA ( 1 ) as a template, extracellular domains (amino acids 1–276) of HLA-G1 were amplified by PCR using primers: 5′-ctcgagcatatgggTtcTcaTtcTatgCgTtatttTagcgcAgcAgtTtcTcgTccAggccgcggg-3′ and 5′-atgcagggatccctgcttccatctcagcatgagggg-3′ and cloned into a pGMT7 vector derivative containing a BirA recognition and biotinylation site in frame at the COOH-terminus ( 11 ). The NH 2 -terminal primer contained several synonymous nucleotide substitutions (capitalized) designed to optimize protein expression from Escherichia coli strain BL21 pLysS. HLA-G tetramers were created essentially as previously described ( 11 ), using synthetic peptide RIIPRHLQL (or KIPAQFYIL where indicated) (Genosys) previously shown to interact with HLA-G ( 31 , 32 ). Dilutions for flow cytometry staining contained ∼14 μg/ml of refolded HLA- G/β2 microglobulin. HLA-E*0101 and HLA-B*2705 tetramers were refolded with peptides VMAPRTLFL and KRWIILGLNK, respectively ( 11 , 33 ). Staining of PBMCs and transfectants was performed using standard protocols. For PBMCs, PBS 0.05% NaN 3 buffer was supplemented with 10% human serum for blocking and primary incubation, and 1–2% human serum for washes and secondary incubations. PBMCs were stained on ice immediately after Ficoll-Hypaque separation or frozen and thawed immediately before use. Cells were analyzed on a FACScan™. We constructed HLA-G tetrameric complexes refolded with a synthetic self-peptide (RIIPRHLQL) derived from human histone H2A ( 31 , 32 ). These PE-labeled HLA-G tetramers were used to stain PBMCs from healthy individuals. No significant HLA-G tetramer binding was observed on CD56 + NK cells, CD3 + T cells, or CD19 + B cells within the gated lymphocyte population . In contrast, when an electronic gate was set on myelomonocytic cells, significant HLA-G tetramer interaction was observed. CD14 high cells, representing the majority of monocytes, stained weakly, with intensity of staining varying between individuals . In addition, a subset of cells within the myelomonocytic population exhibited considerably brighter HLA-G tetramer staining . These cells ranged from CD14 high to CD14 − . In freshly isolated PBMCs from six individuals, this HLA-G Tet bright subset represented 5–12% of cells within the myelomonocytic gate, or 1–2.8% of total PBMCs. Almost indistinguishable patterns of staining were obtained with an HLA-G tetramer refolded with a second peptide (KIPAQFYIL) (data not shown) also known to bind to HLA-G ( 31 ). However, interactions with myelomonocytic cells were not unique to HLA-G, as tetramers of other MHC class I molecules exhibited similar staining, although often with considerably less intensity (data not shown). To further characterize the cells staining intensely with HLA-G tetramers, the expression of a number of other cell surface markers was examined in three individuals. Levels of CD13, CD32 (FcγRII), and CD33 on HLA-G Tet bright cells were comparable or slightly lower than most monocytes . The expression of CD33 and CD13 on the HLA-G Tet bright subset was consistent with these cells having a myeloid origin. The HLA-G Tet bright cells appeared to form a distinct subgroup, expressing much higher CD16 (FcγRIII), lower CD64 (FcγRI), lower CD11b, higher CD11c, higher CD45RA, and slightly lower CD45RO levels than the majority of monocytes . Similarly, HLA-G Tet bright cells showed slightly higher levels of costimulatory CD86 (B7-2) and CD40 molecules and MHC class II (anti–HLA-DR or anti–pan-class II) compared with typical monocytes . This phenotype is very similar to a previously described CD16 + CD14 mid monocyte subset ( 34 ). Ziegler-Heitbrock has suggested that these CD16 + CD14 mid cells may be differentiating to become tissue macrophages ( 34 ). Intracellular staining for CD68, which is highly expressed by macrophages, did reveal a marginally brighter signal in HLA-G Tet bright cells (data not shown). However, the HLA-G Tet bright subset failed to stain with antibodies to scavenger receptor A or mannose receptor found on tissue macrophages (data not shown). Many of these patterns of marker expression are also suggestive of a peripheral blood dendritic cell (DC) phenotype ( 35 – 37 ). Expression of CD16, however, is inconsistent with prior descriptions of blood DCs ( 35 – 37 ). HLA-G Tet bright cells also fail to express DC-associated markers CD1a and CD83 (data not shown). Nonetheless, the HLA-G Tet bright subset could represent a stage in either the macrophage or DC differentiation pathways. To search for the receptors responsible for the observed staining of PBMCs, we stained a number of transfectants with HLA-G tetramers. We began with cells transfected with several NK cell receptors previously suggested to interact with HLA-G ( 6 – 8 , 15 , 16 ). As shown in Fig. 3 A, HLA-G tetramers did not bind to transfectants expressing high levels of CD94/NKG2A or CD94/NKG2C/DAP12. Similarly, we did not observe any binding of HLA-G tetramers to cells transfected with KIR2DL1, KIR2DL3, KIR3DL1, KIR3DL2, or KIR2DL4 members of the KIR family . These findings correlate with the failure of HLA-G tetramers to stain NK cells from peripheral blood. It remains possible that interactions might be missed if they were of low affinity or peptide dependent, or required glycosylation unattained on HLA-G expressed in E . coli . However, the efficient interactions of similarly constructed HLA-E tetramers with CD94/NKG2A and C receptors and HLA-B*2705 tetramers with KIR3DL1 considerably weaken such arguments. In addition, the previously reported recognition of HLA-G by NK clones expressing CD94/NKG2 receptors ( 6 – 8 ) can be explained by interactions with HLA-E, which is upregulated upon acquisition of leader sequence peptides from HLA-G ( 9 – 14 ). We next investigated HLA-G tetramer interactions with receptors of the ILT (or LIR or MIR) family ( 18 – 27 ). HLA-G tetramers efficiently stained transfectants expressing ILT4 and ILT2 receptors . HLA-G tetramer binding, however, was quite dependent upon the density of receptor. Significantly higher expression of ILT2 was necessary to observe efficient HLA-G tetramer binding, suggesting that this interaction may have relatively lower affinity compared with ILT4 . However, such interactions were not unique to HLA-G, as tetrameric complexes of several other MHC class I molecules also stained the ILT2 and ILT4 transfectants (data not shown and reference 20 ). Binding of certain molecules, such as HLA-E, appeared less efficient. These results are consistent with the findings that fusion proteins of ILT2 and ILT4 could interact with cells transfected with certain MHC class I molecules including HLA-G ( 19 , 20 , 25 ). HLA-G tetramers did not bind to cells transfected with ILT1, ILT3, ILT5, or ILT8 , confirming previous reports that failed to observe interactions of fusion proteins with MHC class I molecules ( 20 , 21 , 24 ). To determine if the HLA-G tetramer staining of blood monocytes was the result of interaction with these ILT receptors, we stained PBMCs with HLA-G tetramers in the presence of ILT-reactive mAbs. The addition of 40H2 mAb, recognizing several members of the ILT family, caused an enhancement of HLA-G tetramer binding to both CD14 high and CD14 mid monocyte subsets, providing direct evidence for the involvement of ILT receptors . Bivalent 40H2 rat IgG1 probably cross-linked receptors thus facilitating formation of multivalent tetramer interactions. 28C8 mAb, which reacted only with ILT2 and ILT4 of the panel of cell surface ILT receptors available for the study , almost completely blocked HLA-G tetramer binding to monocytes . Thus, the interactions responsible for monocyte staining were narrowed to ILT2 and ILT4, consistent with the staining on transfectants, although binding to other receptors sharing very similar antigenic determinants could not be completely excluded. Further experiments with 42D1 and 27D6 mAbs, which recognize ILT4 but not ILT2, revealed that the majority of HLA-G tetramer staining of monocytes was the result of binding to ILT4. 42D1 mAb enhanced HLA-G tetramer staining of monocytes and 27D6 mAb almost completely abrogated HLA-G tetramer binding . These results are consistent with the absence of HLA-G tetramer staining on peripheral blood B cells, T cells, and NK cells that express some ILT2 ( 19 , 25 ). Indeed, the pattern of tetramer staining matches the restricted expression of ILT4 on myelomonocytic cells ( 20 , 25 ). In two-color flow cytometry analyses, HLA-G tetramer binding correlated with ILT4 staining (42D1) (data not shown) and CD16 + and CD14 mid monocytes exhibited higher expression of ILT4 compared with typical CD14 high CD16 − monocytes (data not shown and reference 25 ). If HLA-G Tet bright cells are indeed differentiating to become macrophages ( 34 ) or DCs, ILT4 expression may be modulated in preparation for the tissue phenotype. Alternatively, ligation or lack of ligation of ILT4 may be involved in the control of these differentiation pathways. In conclusion, in this study we demonstrate an interaction of HLA-G tetrameric complexes with peripheral blood monocytes that results from binding to ILT4 receptors. We failed to observe any evidence of interaction of HLA-G tetramers with CD94/NKG2 or KIR NK cell receptors. This suggests that a dominant role of HLA-G may be the modulation of monocyte, macrophage, or DC behavior in pregnancy. ILT4 possesses inhibitory ITIM motifs in its cytoplasmic domain, and its ligation can inhibit Ca 2+ fluxes and tyrosine phosphorylation events in myelomonocytic cells in response to several stimulatory signals ( 20 , 25 ). Thus, HLA-G may provide important inhibitory signals capable of modulating antigen presentation, phagocytosis, antibody-dependent cell-mediated cytotoxicity, or cytokine production by the numerous maternal macrophages present at the maternal–fetal tissue interface. Interactions with ILT4 are not unique to HLA-G, however, as tetramers of several classical MHC class I molecules and HLA-E also bound to monocytes and transfectants, although in some cases with considerably lower efficiency. Fetal trophoblasts are deficient in HLA-A and -B classical MHC class I expression ( 4 ). Thus, it is possible that expression of HLA-G on this tissue only replaces the inhibitory signals to macrophages normally provided by classical class I molecules in other tissues in the body. Although the pattern of HLA-G tetramer staining of PBMCs closely mirrored the restricted expression of ILT4 on myelomonocytic cells ( 20 , 25 ), HLA-G tetramers were also able to stain transfected cells expressing very high levels of ILT2 inhibitory receptors. However, transfectants with lower ILT2 expression did not efficiently bind HLA-G tetramers (data not shown). Thus, the failure of HLA-G tetramers to stain B, T, and NK cells, which express some ILT2 ( 19 , 25 ), likely reflects a relatively lower binding affinity compared with ILT4. However, interactions of HLA-G with ILT2 receptors may allow functional inhibition of many cell subsets ( 19 ). Finally, we can not rule out that HLA-G also serves as a restriction element for maternal T cells, because HLA-G tetramers refolded with self-peptides would not be expected to interact with antigen-specific T cell receptors. | Study | biomedical | en | 0.999995 |
10190907 | C57BL/6 mice were obtained from The Jackson Laboratory . Strain 318 TCR-transgenic mice ( 13 ) were provided by Prof. H. Pircher (University of Freiburg, Freiburg, Germany) and B6Aa 0 /Aa 0 (MHC class II −/− ) mice ( 14 ) by Dr. H. Blüthmann (Hoffmann-La Roche, Basel, Switzerland). All mice were bred and maintained at the Biomedical Research Unit of the Wellington School of Medicine. Cultures were in IMDM and additives were as described ( 8 ). The lymphocytic choriomeningitis virus glycoprotein peptide KAVYNFATM (LCMV 33–41 ) was obtained from Chiron Mimotopes. Supernatant from the cell line IL2L6 was used as a source of human rIL-2. Anti–CTLA-4 clone UC10-4F10-11 (provided by Dr. J. Bluestone, University of Chicago, Chicago, IL), anti-CD3 (145-2C11), anti-CD28 (37.51), anti-CD8 (2.43), anti-CD4 (GK1.5), anti-CD11c (N418), anti-FcγRII (2.4G2), anti-Vβ8.1/8.2 (KJ16.133.18), and anti-CD44 (I42/5) were affinity purified from culture supernatants using protein G–Sepharose ( Pharmacia Biotech ) and conjugated to FITC or biotin. Anti-Vα2–PE mAb and streptavidin–Cy-Chrome were obtained from PharMingen . Control IgG was affinity purified from hamster serum using protein G–Sepharose. Cells were stained in PBS containing 2% FCS and 0.01% sodium azide as described ( 15 ). Lymph node cell suspensions were layered onto a Percoll gradient ( Pharmacia Biotech AB ) and the high density cells taken from the interface of 60 and 70% Percoll layers. CD4 + and Ig + cells were depleted by treatment with anti-CD4 mAb followed by anti-Ig magnetic bead adherence ( Dynal A.S.). The remaining cell suspension contained 95% CD8 + T cells, with no detectable CD4 + T cells and <2% B220 + cells. For cross-linking experiments, 10 5 resting CD8 + -enriched cells were cultured with 10 5 polystyrene beads coated with anti-CD3 and either anti–CTLA-4 or control IgG in the presence or absence of soluble anti-CD28 as described ( 16 ). Control cultures were provided with 50 U/ml human rIL-2. Proliferation was determined by 3 H-TdR incorporation over the last 8 h of a 72-h culture. Bone marrow cells from C57BL/6 mice or MHC class II −/− mice were cultured in 20 ng/ml IL-4 and 20 ng/ml GM-CSF for 6–8 d as described ( 17 ). Cultures typically contained 90–100% DC as determined by FACS ® staining with anti-CD11c mAb. DC were loaded with Ag by incubation in medium containing 10 μM LCMV 33–41 for 2 h. Lymph node cell suspensions were prepared from line 318 mice, and the percentage of T cells expressing transgenic TCR was determined by flow cytometry using anti-TCR Vα2 and anti-TCR Vβ8.1/8.2 mAb. The equivalent of 3–5 × 10 6 Vα2 + Vβ8 + T cells were injected intravenously into C57BL/6 recipients, and on the same day, mice were given an intraperitoneal injection of 1 mg anti–CTLA-4 mAb or control IgG. 1 d later, recipients were immunized by subcutaneous injection of 10 5 LCMV 33–41 peptide–loaded DC or untreated DC in IMDM. For each experiment, a group of adoptive transfer recipients was left unmanipulated to serve as a control. For experiments in MHC class II −/− recipients, the donor cell preparations were depleted of CD4 + and Ig + cells as described above. C57BL/6 mice received TCR-transgenic T cells, were treated with anti–CTLA-4 or control IgG, and were immunized with 3 × 10 4 DC as described above. 7 d after DC immunization, splenocytes were harvested, depleted of CD4 + and Ig + cells, and tested for cytotoxic activity in vitro by JAM test on 5,000 labeled EL4 cells that had been incubated in the presence or absence of 1 μM LCMV 33–41 peptide for 1 h at 37°C before the assay ( 18 ). All cultures were performed in triplicate. We used anti–CTLA-4 mAb conjugated to polystyrene beads to examine the effect of CTLA-4 cross-linking on the activation and proliferation of purified resting CD8 + T cells in culture. Lymphocyte preparations from line 318 TCR-transgenic mice were depleted of CD4 + and Ig + cells using Ab-coated magnetic beads. Enriched CD8 + T cells were cultured with beads coated with either anti-CD3 and anti–CTLA-4 or anti-CD3 and control IgG in the presence of a positive costimulatory signal provided by soluble anti-CD28. As shown in Fig. 1 A, after 24 h both control Ab–treated and anti– CTLA-4–treated cultures contained activated CD8 + T cells with markedly increased expression of the activation markers CD25 and CD69 as compared to resting cells. However, whereas expression of these activation markers was maintained until after 48 h in control cultures, it was rapidly lost in the presence of anti–CTLA-4. No increase in cell death was apparent in anti–CTLA-4–treated cultures as compared to control cultures (data not shown). Proliferation of CD8 + T cells in these cultures was assayed 64–72 h after activation . In the presence of anti-CD28, control cultures were highly activated and showed significant levels of proliferation. In contrast, cross-linking of CTLA-4 with mAb-conjugated beads completely inhibited proliferation. The inhibitory function of anti–CTLA-4 was overridden by addition of exogenous IL-2. Therefore, the proliferative function of CD8 + T cells can be directly inhibited by signals mediated via CTLA-4. Similar results have been reported by Walunas et al. using CD8 + T cells from the TCR-transgenic strain 2C ( 19 ). To determine whether CTLA-4 signals regulate the activation of CD8 + T cells in vivo, we examined the effect of a neutralizing anti– CTLA-4 mAb on the accumulation of specific CD8 + T cells in the lymph nodes of mice immunized with Ag-loaded DC. An adoptive transfer model was used ( 15 ) in which nonmanipulated C57BL/6 hosts received 5 × 10 6 TCR-transgenic T cells from strain 318 mice. These T cells are specific for LCMV 33–41 in association with H-2D b and can be identified by their Vα2 + Vβ8 + CD8 + phenotype. Anti– CTLA-4 mAb or control IgG was administered intraperitoneally at the time of adoptive transfer, followed by DC loaded with LCMV 33–41 peptide on day 1. Control animals received either DC that had not been loaded with Ag or no DC immunization at all. Activation and accumulation of Vα2 + Vβ8 + CD8 + T cells in the draining lymph nodes were examined on day 5 after immunization, as preliminary experiments showed that both responses were maximal on this day (data not shown). The data in Fig. 2 are presented as fold increase in the total number of Vα2 + Vβ8 + CD8 + T cells in immunized mice over the number of the same cells in animals that were not immunized. This is because the fold increase in Vα2 + Vβ8 + CD8 + T cells was reproducible in different experiments, whereas the absolute cell number varied. In control IgG–treated animals, an average 2.4-fold increase in the number of Vα2 + Vβ8 + CD8 + T cells was observed in response to Ag-loaded DC, whereas immunization with DC without Ag failed to induce any increase. Significantly, when animals were treated with anti–CTLA-4 mAb, the Ag-induced accumulation was greater, with an average sixfold increase observed. This reflected both an increase in the proportion of Vα2 + Vβ8 + CD8 + T cells and an increase in the total number of lymph node cells (twofold). Immunization with Ag-loaded DC induced increased CD44 expression on a significant proportion of Vα2 + Vβ8 + T cells, and this proportion was greatly increased in animals treated with anti–CTLA-4 mAb . Importantly, increased CD44 expression was strictly Ag dependent and was not detected on cells not expressing the Vα2 + Vβ8 + receptor. Interestingly, a twofold increase in the cellularity of the draining lymph node, with no increase in the percentage of Vα2 + Vβ8 + CD8 + cells, was observed in mice treated with anti–CTLA-4 and immunized with DC only, resulting in an increase in the absolute number of Vα2 + Vβ8 + cells. The increased cellularity was immunization related, as it was not observed in nondraining lymph nodes. No increased expression of CD44 or other activation markers was observed on these Vα2 + Vβ8 + cells (not shown), indicating that blockade of CTLA-4 signals was not sufficient to induce T cell activation in the absence of Ag. Taken together, these results suggest that inhibition of CTLA-4–mediated signaling results in enhanced Ag-specific proliferation of CD8 + T cells in vivo. It was possible that the enhanced CD8 + T cell activation induced by anti–CTLA-4 mAb treatment was due to increased helper function of CD4 + T cells. To examine this possibility, we repeated the DC immunization experiments using MHC class II −/− DC, which are unable to present Ag to CD4 + T cells and elicit T cell help. MHC class II −/− mice were also used as immunization recipients, as in these mice, no T cell help would be available if Ag was transferred from the injected DC to host APC. As shown in Fig. 3 , immunization with MHC class II −/− DC loaded with LCMV 33–41 peptide induced selective accumulation of Vα2 + Vβ8 + CD8 + T cells in the draining lymph nodes of MHC class II +/+ and MHC Class II −/− mice. More importantly, treatment with anti–CTLA-4 mAb significantly enhanced the accumulation of Vα2 + Vβ8 + CD8 + T cells and their expression of CD44 (not shown), regardless of the expression of MHC class II on host APC. Again, as for Fig. 2 , treatment with anti–CTLA-4 caused an increase in the cellularity of the draining lymph node in mice immunized with DC only; however, the percentage of Vα2 + Vβ8 + CD8 + cells was not increased compared to controls nor was their expression of activation markers altered. These results suggest that the enhanced accumulation of Vα2 + Vβ8 + T cells observed with anti–CTLA-4 treatment in DC-immunized mice is not dependent on the provision of CD4 + T cell help. We conclude that CD8 + T cells can be directly regulated in vivo by signals mediated via CTLA-4 molecules. To determine whether in vivo treatment with anti–CTLA-4 mAb affects the effector function of CD8 + T cells, we assayed cytotoxic activity in mAb-treated adoptive transfer recipients that had been immunized with DC. Spleen cells were recovered 7 d after immunization, depleted of CD4 + T cells and Ig + cells, and assayed directly on LCMV 33–41 peptide–coated EL4 targets. As is seen in Fig. 4 , Ag-specific cytotoxicity was barely detectable when CD8 + effector cells were prepared from DC-immunized, control IgG–treated mice. Therefore, the cytotoxic activity induced by DC immunization was below the threshold of detection by this technique. In contrast, in vivo treatment with anti–CTLA-4 mAb resulted in a measurable increase in specific cytotoxicity, with 50% lysis observed at an E/T ratio of 150:1 when MHC class II +/+ DC were used for immunization. When MHC class II −/− DC were used, 35% lysis was observed. Increased cytotoxic activity of effector CD8 + T cells in the anti–CTLA-4–treated groups was apparent even when the E/T ratios were adjusted for the percentage of Vα2 + Vβ8 + cells (data not shown). These results indicate that CTLA-4–mediated signals regulate the cytotoxic activity of CD8 + T cells, also in the absence of CD4 + help. In this paper, we show that CD8 + T cell responses induced by Ag presented on DC are amplified when CTLA-4–dependent signaling is inhibited in vivo. While anti– CTLA-4 mAb treatment was known to enhance several kinds of T cell immune responses ( 6 – 11 ), it had not been previously reported that increased CD8 + T cell activation is also observed when DC are used as APC, a finding that may have significant implications for the use of anti– CTLA-4 in tumor immunotherapy. We investigated whether anti–CTLA-4 enhanced the activity of CD8 + T cells directly or by increasing the availability of CD4 + T cell help. To this purpose, we carried out experiments in which the potential contribution of CD4 + T cells to the CD8 + T cell response was progressively reduced. We observed augmented CD8 + T cell responses regardless of the availability of CD4 + T cells, indicating that CD4 + T cells were not critical to the observed effect. As also reported by Walunas et al. ( 19 ), a purified cell culture system confirmed that CD8 + T cells could respond directly to CTLA-4–mediated signals in vitro. However, although it is clear that the effects of anti–CTLA-4 we have observed are independent of CD4 + T cell help, CD4 + T cells are thought to be necessary for the activation of DC and for the productive development of a CD8 + T cell response ( 20 ). Because we could observe good CD8 + T cell responses even in the absence of CD4 + T cells, we must conclude that our DC were sufficiently activated before injection to be able to induce good CD8 + T cell priming. The findings reported in this paper are relevant to the described enhancement of tumor immunity induced by in vivo treatment with anti–CTLA-4 mAb ( 12 ). Treatment with anti–CTLA-4 mAb in vivo is thought to block the delivery of a negative signal, mediated by B7 ligands on B7-expressing cells. Because tumor cells are generally B7 − , a third cell type must be providing B7 in this system. Bone marrow–derived APC, presumably DC, have been reported to take up and present self Ag ( 21 ) as well as tumor Ag ( 22 ) from peripheral tissues in physiological situations. We thus hypothesized that tumor immunity induced by anti–CTLA-4 treatment is most likely due to enhanced T cell activation by tumor antigen presented by DC. In this paper, we show that both CD8 + T cell activation and specific cytotoxic activity induced by immunization with DC are amplified in the presence of anti–CTLA-4 mAb, possibly resulting in the tumor immunity described by Leach et al. ( 12 ). Our results also imply that tumors in which Ag fails to gain access to a DC, because of either poor antigenicity or limited DC presence in the tumor, may fail to respond to anti–CTLA-4 treatment. Conversely, manipulations which increase local inflammation and therefore access of tumor Ag to DC ( 23 ), or tumor vaccination procedures, could become more effective if used in combination with anti–CTLA-4 treatment ( 24 ). As in the case of CTLA-4–deficient mice ( 25 ), we could observe no spontaneous T cell activation in the absence of Ag when CTLA-4 signaling was blocked. Surprisingly, however, we observed that lymph node cellularity was increased by anti–CTLA-4 treatment even after immunization with DC only. This increase was restricted to the draining lymph nodes, indicating that it was immunization related. DC have been reported to induce Ca 2+ responses in T cells in the absence of Ag ( 26 ). This low level of stimulation may be amplified by the removal of CTLA-4 signals. In conclusion, blockade of CTLA-4 directly enhances the ability of CD8 + T cells to respond to Ag presented on DC, amplifying their expansion and accumulation in lymphoid organs and their cytotoxic activity. This finding may help explain the antitumor effect of anti–CTLA-4 mAb treatment, and, if extended to CD4 + T cells, the autoimmune phenotype observed in CTLA-4–deficient mice. | Study | biomedical | en | 0.999997 |
10190908 | Normal C57BL/6 mice were obtained from The Jackson Laboratory and were designated ZAP +/+ . ZAP-70 knockout mice were provided by Dennis Loh (Roche Research Institute, Nutley, NJ) and Izumi Negishi (Nippon Roche Research Center, Kanagawa, Japan; reference 5 ), were bred in our own animal colony, and were designated ZAP −/− . F1 offspring between B6 and ZAP-70 knockout mice were designated ZAP +/− . ST mice are homozygous for a spontaneously arising point mutation in ZAP-70's kinase domain, which renders them kinase dead ( 6 ). Double knockout mice lacking both ZAP-70 and MHC II expression were generated by crossing ZAP-70 knockout and MHC II knockout mice together, and screening the F2 generation for animals designated as ZAP −/− II −/− . The care of experimental animals was in accordance with National Institutes of Health guidelines. Upon their removal from the thymus, thymocytes were kept strictly at 4°C in all experiments unless otherwise indicated, in order to avoid the biochemical alterations that occur in DP thymocytes upon removal from their intrathymic signaling environment ( 3 , 8 ). DP thymocyte populations (>96% pure) were obtained by panning whole thymocytes on anti-CD8 plates and collecting the adherent fraction. Antibodies used for immunoprecipitation and/or immunoblotting in this study were specific for ζ (serum No. 551), ZAP-70, CD4 (RM4.5, PharMingen ), phosphotyrosine (4G10, Upstate Biotechnology, Inc.), or lck (serum No. 688). Biotinylated antibodies used for TCR and coreceptor cross-linking were specific for TCR-β (H57-597) or CD4 (GK1.5). DP thymocytes were coated with biotinylated anti-TCR and/or biotinylated anti-CD4 mAbs for 10 min at 4°C, after which the cells were warmed to 37°C and exposed to streptavidin for the indicated time (usually 5 min). Where indicated, cells were also treated with pervanadate (0.3 mM H 2 O 2 and 0.1 mM Na 3 VO 4 ) for 5 min at 37°C ( 2 , 3 ). DP thymocytes were lysed in 1% triton and the lysates immunoprecipitated with the indicated antibodies and resolved by SDS-page under reducing conditions (5 × 10 7 cells per sample). The gels were transferred onto immobilon PVDF membranes ( Millipore ), blotted with the indicated antibodies, and visualized by chemiluminescence. DP thymocytes were lysed at 10 8 cells/ml in lysis buffer containing 1 mM vanadate (a potent inhibitor of protein tyrosine phosphatases) and 1% Triton X-100; and the lysates immunoprecipitated with the indicated antibodies. Immune complexes were incubated at ambient temperature for 3 min in kinase buffer containing 15 μCi/sample of γ-[ 32 P]ATP, after which the immune complexes were resolved by SDS-PAGE and visualized by autoradiography. Radiolabeled proteins in the immune complex kinase assay reflect transfer of 32 P by an activated PTK molecule present in the immunoprecipitate ( 3 ). Greater than 50% of surface TCR complexes on immature DP thymocytes in the thymic cortex contain ‘constitutively' tyrosine phosphorylated ζ ITAMs ( 3 , 9 ), in contrast to <5% of TCR on mature T cells in the periphery ( 3 , 10 ). Constitutive ITAM phosphorylation in DP thymocytes results from lck signals generated by interactions between DP thymocytes and thymic cortical epithelium that are mediated primarily, but not exclusively, by CD4–MHC II interactions. Aggregation of surface CD4 molecules on DP thymocytes by engagement of MHC II on cortical thymic epithelium activates CD4-associated lck to phosphorylate ζ ITAMs, after which the activated lck molecules are degraded ( 2 , 3 , 8 – 11 ). The tyrosine phosphorylated ITAMs then recruit ZAP-70 molecules that remain enzymatically inactive ( 3 , 10 ), perhaps because the remaining pool of activated lck available to the TCR in DP thymocytes is insufficient to induce ZAP-70 activation. Importantly, the lck that is available to the TCR complex in DP thymocytes appears to be primarily the lck that is associated with coreceptor molecules and for which coreceptor molecules compete for binding ( 2 , 3 ). However, additional factors may also influence the availability of lck to the TCR. For example, transfection experiments in nonlymphoid cells have found that ITAM phosphorylation by lck is increased by ZAP-70, an effect ascribed to ZAP-70's protection of phospho-ITAMs from dephosphorylation ( 12 ). To assess a possible role for ZAP-70 in ITAM phosphorylation in DP thymocytes, we examined purified DP thymocyte populations from mice expressing different quantities of ZAP-70. Remarkably, we found that the extent of ζ phosphorylation was proportional to the amount of ZAP-70 protein expressed . Importantly, all three DP thymocyte populations (ZAP +/+ , ZAP +/− , and ZAP −/− ) contained comparable levels of ζ protein . To determine if the relationship between ZAP-70 expression and ζ phosphorylation required ZAP-70 kinase activity, we examined ζ phosphorylation in DP thymocytes from mutant ST mice that express only kinase-dead ZAP-70 proteins. Mutant ZAP-70 proteins are less stable than their wild-type counterparts so that DP thymocytes from mutant ST mice contained reduced steady state levels of ZAP-70 protein, comparable with the levels of ZAP-70 protein expressed in ZAP +/− DP . Interestingly, tyrosine phosphorylation of ζ ITAMs from mutant ST and ZAP +/− mice were also essentially equivalent , especially considering that the ST lane contained somewhat less ζ protein. Thus, ST thymocytes contained phosphorylated ζ ITAMs in amounts that were roughly concordant with their reduced level of ZAP-70 protein, indicating that ζ ITAM phosphorylation is quantitatively dependent upon ZAP-70 protein but is independent of ZAP-70's enzymatic activity. We next wished to determine if the requirement for ZAP-70 in coreceptor-induced ITAM phosphorylation could be overcome by coengagement of CD4 with the TCR. Although TCR plus CD4 co-engagement did induce ζ ITAM phosphorylation in DP thymocytes from ZAP +/+ mice, it failed to do so in DP thymocytes from ZAP −/− mice . The intrinsic ability of ζ ITAMs to be tyrosine phosphorylated was not obviously altered in ZAP −/− thymocytes, as indicated by treatment of DP thymocytes with pervanadate, which inhibits intracellular tyrosine phosphatases and so indiscriminately activates intracellular PTK ( 13 ). To further examine TCR ITAM phosphorylation in TCR plus CD4–signaled DP thymocytes, we assessed DP thymocyte lysates in an immune complex kinase assay for their ability to tyrosine phosphorylate TCR ITAMs in vitro . Concordant with our observation in intact DP thymocytes, in vitro phosphorylation of TCR ITAMs (ε and ζ) was markedly reduced >10-fold in ZAP −/− relative to ZAP +/+ anti-ζ immunoprecipitates from TCR plus CD4–signaled DP thymocytes . Because dephosphorylation of TCR ITAMs is already inhibited in in vitro kinase assays by exogenously added vanadate, a potent tyrosine phosphatase inhibitor, the requirement for ZAP-70 protein in ITAM phosphorylation indicates that ZAP-70's role is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this experiment reveals that ZAP-70 promotes tyrosine phosphorylation of TCR ITAMs in DP thymocytes. Because ITAM phosphorylation is mediated primarily by lck, we next considered that in vivo ITAM phosphorylation might be less dependent upon ZAP-70 in DP thymocytes containing increased amounts of TCR-accessible lck. Consequently, we examined signaling in DP thymocytes that lacked ZAP-70 but contained increased CD4-associated lck, because their thymi did not express MHC II . In the absence of MHC II engagement in the thymus, CD4 molecules on DP thymocytes are not aggregated and, consequently, are not depleted of associated lck molecules ( 2 ). Indeed, ZAP −/− DP thymocytes from II −/− mice contained more CD4-associated lck than did ZAP −/− DP thymocytes from II +/+ mice . More importantly, cocross-linking of TCR plus CD4 surface molecules on DP thymocytes from ZAP −/− II −/− mice induced significant ITAM phosphorylation despite the absence of ZAP-70 protein , indicating that the requirement for ZAP-70 diminishes with increasing amounts of coreceptor-associated lck. The pool of CD4-associated lck in DP thymocytes can also be increased by removing thymocytes from their thymic microenvironment and culturing them as single cell suspensions in the absence of MHC II + cells. In such cultures, intrathymic engagements are disrupted, permitting newly synthesized CD4 and lck molecules to repopulate the depleted pool of coreceptor-associated lck ( 2 ). It is clear that TCR plus CD4 cocross-linking increased ITAM phosphorylation in these cultured DP thymocytes whether or not ZAP-70 protein was present . Thus, ITAM phosphorylation is less dependent upon ZAP-70 in DP thymocytes containing increased amounts of coreceptor-associated lck. A more stringent assessment of the role of ZAP-70 in regulating ITAM phosphorylation is the one signaled by engagement of the TCR alone, as opposed to ITAM phosphorylation signaled by the forcible coengagement of TCR with CD4 . Signaling by isolated TCR cross-linking requires that lck be captured within TCR aggregates, which is an inefficient method of bringing lck into contact with TCR ITAMs compared with direct TCR plus CD4 cocross-linking. Nevertheless, isolated TCR cross-linking did signal ITAM phosphorylation in cultured DP thymocytes that contained ZAP-70 protein (even enzymatically inactive ZAP-70 protein in mutant ST thymocytes), but failed to signal ζ ITAM phosphorylation in cultured DP thymocytes that lacked ZAP-70 protein . The inability of isolated TCR cross-linking to induce ITAM phosphorylation in cultured DP thymocytes lacking ZAP-70 protein indicates that ZAP-70 is specifically required for ITAM phosphorylation when the amount of lck available to TCR aggregates is limiting. Current models of TCR signaling are derived primarily from experiments in mature T cells and cell lines, and do not include a role for ZAP-70 in early TCR signaling events such as ITAM phosphorylation ( 7 ). In contrast, our study found that in immature DP thymocytes ZAP-70 protein is required for ITAM phosphorylation, and that the requirement for ZAP-70 protein decreases as the amount of lck available to the TCR quantitatively increases. ZAP-70's role in ITAM phosphorylation in DP thymocytes was not limited to protecting phosphorylated ITAMs from dephosphorylation, as ZAP-70 was required for in vitro ITAM phosphorylation by DP thymocyte lysates even though tyrosine dephosphorylation was already inhibited by the pharmacologic agent vanadate. Rather, our study indicates that ZAP-70 in DP thymocytes promotes lck's phosphorylation of TCR ITAMs, a function that is especially significant in immature DP thymocytes with limiting lck. Our understanding of ZAP-70's role in promoting ITAM phosphorylation by lck in DP thymocytes builds on the key finding that lck can bind to ZAP-70 via an SH2 domain interaction ( 14 , 15 ). We suggest that ITAM phosphorylation in DP thymocytes occurs in two phases. In the first phase of ITAM phosphorylation, a chance encounter between an activated lck molecule and a TCR ITAM induces initial ITAM phosphorylation without any involvement of ZAP-70. The number of TCR ITAMs initially phosphorylated by such chance encounters depends upon the number of activated lck molecules available to the TCR. In immature DP thymocytes, the amount of lck available to the TCR is limiting, so that only a few TCR ITAMs are phosphorylated in this initial ZAP-70–independent phase, perhaps only a single ITAM in a TCR aggregate. In the second phase of ITAM phosphorylation, the initially phosphorylated ITAM is bound by a ZAP-70 molecule whose backbone tyrosine residue is phosphorylated and then bound by activated lck. The activated ZAP-70–bound lck molecule is now within the TCR complex and so can efficiently phosphorylate the remaining ITAMs in the TCR aggregate. (In fact, being bound to ZAP-70 via its SH2 domain might prolong lck's enzymatic activity because lck's SH2 domain would not be available to bind lck's regulatory phosphotyrosine, interfering with downregulation of lck's kinase activity.) The newly phosphorylated ITAMs are subsequently bound by additional ZAP-70 molecules and it is these additional ZAP-70 molecules that make up the majority of ZAP-70 molecules constitutively associated with TCR in DP thymocytes. These ZAP-70 molecules are only recruited into the TCR complex after the second phase of ITAM phosphorylation and they are not themselves tyrosine phosphorylated in DP thymocytes, presumably because the activated lck has already disappeared from the TCR complex. However, these TCR-bound ZAP-70 molecules can be efficiently activated by subsequent TCR plus coreceptor engagements of intrathymic ligands that bring newly activated lck molecules into the TCR complex. Thus, ZAP-70's promotion of ITAM phosphorylation represents a signal amplification mechanism in DP thymocytes. In conclusion, our study demonstrates that ZAP-70 promotes ITAM phosphorylation in DP thymocytes in response to TCR and/or coreceptor engagement, and that DP thymocytes are particularly dependent upon ZAP-70 for ITAM phosphorylation because their pool of available lck is limiting. | Study | biomedical | en | 0.999997 |
10209018 | All K . lactis haploid strains were derived from the parental haploid strain K7B520 that has been previously described . K7B520 was transformed with up to 10–50 ng/μl of pTER-BX plasmid, a YIP5 derivative containing the wild-type TER1 gene with described template mutations . Cells were electroporated (1.5 kV, 200 Ω, 25 μF) and plated on medium lacking uracil and containing 1 M sorbitol. After 2 d, transformants were restreaked onto 5-fluoroorotic acid–containing medium and resistant colonies were screened for the desired gene replacement by digestion of PCR products with the restriction enzyme whose site was formed in the mutated template. PCR primers used were: upper strand, 5′ GTC AAG TTC TGG AGG and lower strand, 5′ CGA AGA GAA GAA TGG ( GIBCO BRL ). Cells were passaged by restreaking representative colonies onto yeast extract/peptone/dextrose and grown for 3 d at 30°C. Genomic DNA was prepared from cells grown in yeast extract/peptone/ dextrose at 30°C until late log phase. At least three independent isolates were cultured for each of the uncapped ter1-Acc , ter1-Bgl , and ter1-Kpn strains. At least three independent loop-outs were cultured for ter1-Bcl recapped and Δter1 strains. DNA was cut with EcoR1 ( New England Biolabs Inc. ) and the appropriate second restriction enzyme at 37°C overnight (47°C for BclI ) and electrophoresed 0.8% agarose, 1× TBE gels at 40 V for 24 h or 1% 0.5× TBE pulsed-field gels at 230 V for 16 h (50 ms pulse time). After depurination, samples were transferred to Hybond N + nitrocellulose ( Amersham Corp. ) and cross-linked with 1200 μJ using a Stratalinker 1800 (Stratagene Inc.). Hybridizations were carried out in 0.5 M Na 2 HPO 4 , 7% SDS, 0.5 mM EDTA at 50°C for 4–16 h and washed twice for 5 min at 40–50°C with 200 mM NaCl wash buffer. The wild-type (WT) 1 telomeric sequence probe used for all blots shown was 5′ TAA TCA AAT CCG TAC ACC ACA TAC C. Blots were exposed to autoradiography film ( Eastman-Kodak Co. ) and scanned at 300 dots-per-inch resolution. Triplicate K . lactis cultures were grown to an A 600 of 0.5–1. Triplicate cultures from a single stock were used for each uncapped strain, while three independent loop-out strains were used for re-capped and Δter1 strains. Cells were centrifuged at ∼5,000 rpm in a clinical centrifuge and washed twice in 1 ml PBS. Washed cells were then fixed in 70% ethanol in PBS and diluted to 1 A 600 U/ml (∼2 × 10 7 cells/ml). A 1-ml sample of fixed cells was washed twice with 1 ml PBS. Cells were resuspended in 500 μl PBS + 1 mg/ml RNase A (QIAGEN Inc.) and incubated overnight on a rotating platform at 4°C. Afterwards, 20 μg of Proteinase K ( Boehringer-Mannheim Biochemicals ) was added and samples were incubated at 55°C for 1 h. Cells were centrifuged at 5,000 rpm in a microfuge, washed once with 1 ml of PBS, and resuspended in 500 μl of 50 μg/μl propidium iodide ( Sigma Chemical Co. ) for 1 h at room temperature in the dark. Stained samples were diluted 1:10 in PBS and 30,000 ungated events were counted at 650 nm wavelength on a FacsCalibur ® ( Becton Dickinson & Co.). For statistical analyses, the mean of the 2 N peak was measured and the >2.5 N cutoff was calculated using gated plots for each sample. We arbitrarily chose “greater than diploid content” as exceeding 2.5 N. Statistical t-tests were performed using Microsoft Excel 98. Gated histograms were imported into Adobe Photoshop, where line thickness and gray shade were manipulated for overlays . The same fixed samples used for FACS ® analyses (see above) were used for microscopic analyses carried out in parallel. Approximately 1 ml of 70% ethanol/PBS-fixed cells was centrifuged at 5,000 rpm in a microfuge and washed twice with 500 μl of PBS. Cells were stained for 5–15 min at room temperature with 1 μg/ml 2,6–diamidinophenylindole (DAPI) and washed twice with 1 ml of PBS. At least three isolates of each strain were resuspended in 500 μl of PBS and sonicated for 10–30 s at 30% duty with a sonifier (Branson Ultrasonics Corp.). Microscopy was performed using 2 μl of cells on a Microphot FXA microscope ( Nikon Inc. ) at 100× magnification and photographed onto 400 ISO film ( Eastman-Kodak Co. ). The Δter1 image was acquired on a DMLB microscope (Leica Inc.) with a 300 dpi color CCD camera. The total number of cells counted for each mutant is noted in Table I . Photographs were scanned at 300 dpi resolution and then cropped in Adobe Photoshop. The telomeric DNA phenotypes of ter1-Acc , ter1-Bgl , and ter1-Kpn mutants have been reported previously . For the present studies, these findings were confirmed using fresh cultures of the same strains and are summarized in Fig. 1 b. All three mutations resulted in dramatic telomere deregulation and elongation compared with wild-type . We define deregulation as the smeary, heterogeneous population of telomeric DNA species identified on these Southern blots. The apparent sizes of the heterogeneous telomeres in these mutant strains ranged from less than the smallest wild-type telomeric restriction fragment to ≥25 kb. The elongated mutant DNA was largely made up of mutant telomeric DNA repeats, as shown by secondary digestion with each restriction enzyme whose site was copied into telomeric DNA by the mutant telomerase . After cleaving off the mutant repeats, the length of these secondarily digested telomeric fragments reflects the remaining length of the original WT repeat tract that is located internally to the added mutant repeats on the telomere . The size ranges of these internal wild-type repeat tracts were generally similar in all the mutants studied . While the range of internal telomere sizes in ter1 template mutants was comparable to WT, the individual telomere lengths were slightly shorter than WT after the deregulation following uncapping . In addition, the patterns were different from the WT patterns. This change in the patterns of telomeres has been shown previously to be due to the extensive subtelomeric recombination and has been documented in ter1-Acc and late passage Bgl and Kpn strains . The 3.5-kb telomeric fragment that lacks subtelomeric homology to the other telomeres does not appear to participate in these recombination events . Since the telomeres in these strains are more recombinogenic than WT, individual lines passaged by serial restreaking of single colonies have telomeres patterns that are distinct both from other clonal lineages and the same lineage analyzed at different time points . In an extreme case, ter1-Bgl recombined all of its homologous subtelomeres into one species . The telomeres of strains that had their TER1 genes deleted for ∼50 generations ( Δter1 ) were homogeneous and slightly shorter than WT . In the subset of Δter1 cells that survived senescence, the telomere patterns were also significantly altered . In summary, while the telomeres of ter1 template mutants were mostly deregulated and elongated, those of Δter1 survivors were quite short and formed discrete size classes that were regulated. Thus, these short Δter1 telomeres were distinct from the degraded telomeric species observed in the three ter1 template mutant strains. We used FACS ® analysis to investigate the cellular DNA content of ter1 template mutant and Δter1 cell populations . In the WT K . lactis control strain, 13% of the cells contained DNA in excess of diploid content . Similarly, 10–16% of early passage (i.e., ∼150 generation) ter1-Bgl and ter1-Kpn cells, which have short well-regulated telomeres , had greater than diploid DNA content (data not shown). While presenescent Δter1 cells had a DNA content profile similar to WT (data not shown), postsenescent Δter1 survivor cultures with short, relatively homogeneous telomeres exhibited a 27% subpopulation of cells with greater than diploid DNA content . Likewise, ter1-Acc cells, and late passage (>750 generations) ter1-Bgl and late ter1-Kpn cells, which all had deregulated telomeres, showed 27, 35, and 19% subpopulations of cells with greater than diploid DNA content, respectively . While the Acc and late passage Bgl mutants were significantly different from WT (both P < 0.01), the variability of the WT slightly decreased the significance of the difference from the late passage Kpn mutant ( P = 0.06). The increase in DNA content in the ter1 template sequence mutants and Δter1 survivor strains reproducibly coincided with a decreased percentage of cells with 1 N DNA content . These DNA content changes are unlikely to be explained by an increase in telomeric DNA alone, since the Δter1 survivors had much less telomeric signal than the ter1 mutants but still exhibited an increased DNA content . Furthermore, even assuming that all 12 telomeres in haploid K . lactis lengthened to an average of 100 kb, this would only represent approximately one tenth of the haploid genome size . Hence, the increased DNA content of these ter1 mutants was likely to have resulted from either endoreduplication and/or defects in chromosome segregation. To determine whether DNA segregation was affected in ter1 template sequence mutants, we used fluorescence light microscopy and DAPI staining to examine the cellular DNA, and brightfield microscopy to examine overall cell morphology and cell budding indices . We predicted that if DNA segregation were affected, then multiple or large DAPI-staining structures should be visible in a single cell body and some percentage of cells might contain little or no DNA. While WT and presenescent Δter1 cells looked indistinguishable , postsenescent Δter1 survivors had a 4% population of somewhat enlarged, misshapen cells with abnormally distributed DNA (Table I ). These cells also had very degraded cell walls and collapsed buds, as judged by brightfield microscopy . We found that 10% of ter1-Acc mutant cells had cellular defects. These were distinctly different from, and more severe than, the most extreme morphological defects of postsenescent Δter1 cells. Many ter1-Acc mutant cells had multiple DAPI-staining structures , while others had no brightly staining DAPI structures but did contain large areas of diffuse DAPI staining . These Acc cells often appeared to have budding and division defects. They were frequently grossly enlarged or elongated , and some cells formed chains that were resistant to extensive sonication . Other cells were spherical but enlarged to up to five times the diameter of wild-type cells . We use the term “monster cells” generally to describe these phenotypes, with a given cell needing only to exhibit one of these traits to qualify as a monster cell. The ter1-Bgl and ter1-Kpn mutations also resulted in monster cell phenotypes, but only in cell populations with deregulated, elongated telomeres. Thus, early passage (∼150 generations) ter1-Bgl and ter1-Kpn strains with short, regulated telomeres showed no significant monster cell phenotypes above background levels , while isogenic isolates passaged for >750 generations and, with deregulated telomeres, exhibited high levels of severe monster cell phenotypes. The percentages of monster cells in the populations of late passage ter1-Bgl and ter1-Kpn cell strains were 12 and 13%, respectively (Table I ). The same monster cell populations also contained either multiple DAPI-staining structures or decondensed DAPI-staining material and apparent budding and division defects similar to the ter1-Acc mutant cells . Our microscopic analyses highlight the differences between the abnormal phenotypes associated with senescence and monster cells. Although postsenescent Δter1 survivors were phenotypically abnormal, they had irregular, degraded cell walls and collapsed buds unlike those of the ter1 template sequence mutants. Furthermore, postsenescent Δter1 survivors did not exhibit multiple DAPI-staining structures within one cell. Finally, the incidence of monster cells in postsenescent Δter1 populations was at most a third that of other ter1 strains and was considerably more variable between lineages (Table I ). In summary, we concluded that telomere uncapping was caused by the Acc , Bgl , or Kpn mutations and resulted in telomere deregulation and elongation. This correlated with a subpopulation of cells containing DNA in excess of diploid amounts and a significantly increased percentage of morphologically aberrant monster cells that were distinct from postsenescent Δter1 survivors. The multiple DAPI-staining structures in all three uncapped ter1 template mutant strains suggested that the cell's ability to segregate DNA was inversely correlated with the extent of telomere deregulation/elongation. We wished to dissect which property of the uncapped telomeres caused the extreme monster phenotypes described above: deregulation or extreme length. To address this issue, we replaced the mutant ter1 gene in Acc , Bgl , and Kpn strains with a ter1-Bcl allele, which adds phenotypically silent, functionally wild-type, repeats to the telomeric DNA end . The Bcl repeats contain a BclI restriction enzyme site, so that these added marked repeats can be distinguished from preexisting wild-type or other mutant repeats. The Bcl repeats bind Rap1p normally in vitro and thus were predicted to allow the previously disrupted telomere cap to reform at the distal end of the telomere. In all three mutant ter1 strains studied, recapping with Bcl repeats caused a transition from a deregulated smear of telomeric DNA to a series of discrete, length-regulated but still elongated telomeric bands . This transition occurred within ∼50 generations (the earliest time point at which DNA could be analyzed). These reregulated telomeres remained much longer than wild-type , with a significant fraction of the telomere signal still at limit mobility (≥25 kb) for the Bgl and Kpn mutants . Recapping did not significantly change the sizes of the internal wild-type repeat tracts . Digestion of the cap repeats with BclI revealed that only three to four ter1-Bcl repeats were added to each telomere . Interestingly, in late ter1-Bgl cells, the Bcl repeats seemed to incorporate further into some late passage telomeres, since digestion of the cap resulted in large decreases in the sizes of some telomere restriction fragments . The inward migration of these repeats may have been due to faster terminal repeat turnover or recombination, since isogenic cells passaged for an additional 150 generations exhibited a significantly altered telomere pattern . To determine whether telomeric DNA shortened overall after recapping with Bcl repeats, we performed quantitative analyses of the total telomeric hybridization signal in uncapped and recapped lanes, for all three ter1 template mutants . We repeated these analyses using pulse-field gel electrophoresis and compared the total telomeric signals on four chromosomes between uncapped and recapped strains (data not shown). In all cases, there was no significant decrease in telomeric signal after recapping. In summary, the internal WT repeat tracts of uncapped telomeres in ter1 template mutants shortened only slightly, and were longer than those in postsenescent Δter1 survivors. Recapping added an average of three to four ter1-Bcl repeats to the distal tips of telomeres, although in some cases recombination events allowed migration of Bcl repeats further into the telomere. In all cases, however, the recapped ter1 strains regained their ability to regulate telomere length about a new mean size, and the majority of telomeres remained significantly elongated. The recapped ter1 template mutant strains were examined by FACS ® analysis . After recapping, all three ter1 strains eventually exhibited significantly fewer cells with greater than diploid DNA content. The percentage of recapped ter1-Acc and ter1-Bgl cells with greater than diploid DNA content was the same (7%) as in recapped wild-type cells . Interestingly, in ter1-Bgl strains, DNA content did not show an immediate large decrease upon recapping (data not shown). However, ∼150 generations after recapping, the fraction of cells with greater than diploid DNA content was reduced to wild-type levels . In contrast, recapped late passage ter1-Kpn strains showed a significant decrease in cells with greater than diploid DNA content as soon as cells could be analyzed (from 19 to 9%; P < 0.001, data not shown). By the criteria of DAPI staining and brightfield microscopic analyses, the nuclear and cell morphologies of recapped ter1-Acc and late passage ter1-Kpn strains were indistinguishable from wild-type , even though their telomeres remained very long. The early passage recapped ter1-Bgl and ter1-Kpn strains also had DNA contents and percentages of monster cells comparable to wild-type . Immediately after recapping, the late passage ter1-Bgl strain still exhibited a 9% subpopulation of monster cells (Table I ). Qualitatively, these recapped ter1-Bgl cells were not as large or grotesquely malformed as the uncapped ter1-Bgl monster cells . However, ∼150 generations after recapping, the percentage of monster cells returned to wild-type levels even though the telomeres in these cells appeared qualitatively similar to those immediately after recapping . Thus, while Southern blot analyses showed that ter1-Bcl repeats had been physically added to the distal ends of telomeres within 50 generations, it took additional time for late passage ter1-Bgl mutants to establish a cell population with functional telomeric caps. The DNA–protein complex at the end of telomeres is thought to be important for their chromosome-protective functions. When this distal cap complex is disrupted by adding mutant repeats or shortening the existing telomere beyond a critical length, the chromosome becomes uncapped and subject to damage. Uncapping can be defined as the loss of end protection and results in either net telomere shortening or lengthening, increased recombination in telomeric regions, and/or the loss of regulation about a mean telomere length. Here we have addressed two questions related to telomere length regulation in K . lactis . First, what are the cellular phenotypic consequences of uncapped telomeres in ter1 template sequence mutants and postsenescent Δter1 survivor strains? Second, upon finding that cells respond poorly to telomere uncapping, we asked whether it is the resulting telomere deregulation, as opposed to elongation per se, that is correlated with the observed phenotypes. This is the first detailed report in yeast of the cellular morphological consequences caused by telomere uncapping. Telomere uncapping in ter1 template sequence mutants was correlated with a greater than diploid DNA content, aberrant nuclear morphologies, and apparent cell division defects. We conclude that it is the deregulation of telomeres resulting from uncapping, rather than their elongation, that is associated with these phenotypes. The addition of a few wild-type–like repeats to the extreme terminus of the elongated mutant ter1 telomeres allowed strains to regain their ability to regulate telomeres, even though the telomeres were up to 100× longer than wild type. Interestingly, the ter1-Bgl mutant telomeres were not fully capped at first and Bcl repeats migrated further into the telomeres than in other mutants. This may have been due to continued degradative shortening of the telomeres followed by de novo Bcl addition or recombination of the Bcl cap with the internal tracts. However, after being recapped for ∼150 generations, Bgl mutant strains behaved similarly to the Acc and Kpn mutants. Hence, telomere recapping eventually caused the DNA content and cellular morphology to return to normal in all three ter1 mutants. The mechanism by which the deregulation of uncapped telomeres leads to monster cell formation in K . lactis is not known. While general genomic instability and consequent misregulation of gene expression may result in monster cells, the addition of a wild-type telomeric cap is sufficient for recovery of the cell population. In S . cerevisiae , senescing cells show increased chromosome loss . Likewise, elongated, poorly regulated telomeres can increase chromosome loss rates . Telomere uncapping can lead to either telomere shortening ( Δter1 ) or deregulation/elongation ( ter1 template mutants); we have shown here that each has distinct telomere and monster cell phenotypes. The Δter1 survivors had cell walls that appeared degraded and they did not show multiple DAPI-staining structures in one cell body. On the other hand, monster cells of ter1 template sequence mutants had healthy-looking cell walls, decondensed chromatin, often up to 10 nucleus-sized DAPI-staining objects in a single cell body, with frequently no DNA in the adjacent body. Evidence supporting DNA segregation or replication defects includes the observation that the DNA content of cultures with elongated, uncapped telomeres was greatly increased. Taken together with the observation of cells with either increased DAPI- staining or no staining and the morphological results, these results strongly suggest that deregulated telomeres can cause DNA missegregation. We propose the following model for how uncapped telomeres may negatively affect cells . While deletion of ter1 results in telomere shortening until the cap is lost, addition of certain mutant repeats can disrupt the cap without telomere shortening. Mutant repeats that cannot bind Rap1p (i.e., Acc ) result in immediate telomere uncapping, while mutant repeats that retain Rap1p binding ( Bgl and Kpn ) do not result in immediate uncapping. The effects of the Bgl and Kpn mutations accumulate over time until some as yet undefined change(s) in the properties of the Rap1p-nucleated complex on the mutant telomeric DNA prevents functional end protection. Uncapped telomeres may over elongate by telomerase-mediated or recombination pathways at this point . Such telomeres are also subject to degradation, as shown by the smear of telomeric signal migrating faster than wild-type telomeres . Uncapped, elongated telomeres may be recognized as damage, causing cell cycle delay or accidental repair/telomeric fusion, resulting in dicentric chromosome formation. Individual chromosomes or whole genomes may be lost or missegregated. This genomic instability results in further negative phenotypic consequences for the cell. Once polyploidy or aneuploidy occurs, strong selection pressures exist for the healthiest cells, suggesting why the majority of cells in a population are not visually aberrant. However, microcolony analyses of phenotypically wild-type ter1-Acc mutant cells revealed that they continually give rise to subpopulations of monster cells (data not shown). Recapping reverses the phenotypic effects of telomere deregulation. Reestablishment of a functional cap may occur immediately for the population, as in the cases of the recapped ter1-Acc and ter1-Kpn strains, or be slower, as in the case of the late passage ter1-Bgl mutant. We propose that recapping involves reforming a stable DNA–protein complex at the telomere ends, preventing these chromosomes from becoming deregulated and exerting detrimental effects. Cells with stably capped telomeres are likely to have a substantial growth advantage, and once a ter1 population is recapped the frequency of unstable monster cells decreases as healthy cells take over the population. The addition of three to four ter1-Bcl repeats to the termini of the telomere was sufficient to eventually cap ter1-Acc , Bgl , and Kpn mutant telomeres. The relatively few Bcl repeats that migrated into the Bgl telomeres did not appear to have a significant effect on the eventual capping of these telomeres. The telomeres in these recapped strains contain three distinguishable, possibly functional domains: the remaining ∼250–300-bp internal tract of original wild-type repeats most proximal to the centromere, the adjacent large tract of Acc , Bgl , or Kpn mutant repeats, which may exceed 25 kb in length, and the (usually) three to four functionally wild-type ter1-Bcl repeats at the very terminus of the telomere . Whether the remaining internal wild-type repeats were necessary for the reestablishment of a normal cell population after recapping is unknown. Notably, the total telomeric DNA hybridization signal in elongated ter1 mutants remained unchanged after recapping, providing evidence that recapping is not obligatorily associated with a reduction in mean telomere length. This evidence strongly suggests that it is not telomere length, but the very terminal repeats that are important for preventing monster cell formation. It is thought that functionally wild-type telomeres assume a higher-order structure nucleated on Rap1p that protects the chromosome end. The COOH terminus of Rap1p interacts with the Sir and Rif 1 and 2 proteins. Generally, mutations that prevent Rap1p interaction with telomeric DNA (i.e., template mutations), Sirs, and/or Rifs, or COOH-terminal mutations in Rap1p, result in telomere elongation, suggesting that these interactions help stabilize the telomeric complex that regulates telomere length . The results reported here also address whether the monster cell phenotypes observed are the pleiotropic effects of changing the amounts of Rap1p or associated factors in the cell. In the case of the Acc mutation, Rap1p binds with significantly lowered affinity in vitro , and, therefore, Rap1p occupancy of these repeats in vivo may be low. Although 100-fold less Rap1p is predicted to bind Acc repeats, up to 100× as many repeats may be present at each telomere in ter1-Acc strains. Therefore, the overall Rap1p levels at telomeres may not differ greatly between wild-type and ter1-Acc cells. Nevertheless, the structure of their telomeric complexes are likely distinct. In contrast, both Bgl and Kpn mutant repeats have normal Rap1p binding affinity in vitro and upon elongation could potentially titrate Rap1p, along with interacting proteins, away from the scores of genes they regulate. Yet the functionally recapped ter1-Acc , ter1-Bgl , and ter1-Kpn strains all have as much telomeric DNA as uncapped strains and appear as healthy as wild type. Therefore, it is unlikely that titration of Rap1p explains the phenotypes associated with monster cells. It is possible that the uncapped ter1 mutants are unable to regulate the single-stranded ends of the telomere and are therefore unable to regulate length. The Acc , Bgl , and Kpn mutations may affect the interaction of putative end-binding factors, such as K . lactis homologues of the Cdc13p, Est1p, or Stn1p proteins found in S . cerevisiae . If these ter1 mutant repeats were incapable of binding such end factors normally, this could expose the terminal region of the telomere to factors such as recombination-associated activities, including degradation enzymes. A functional cap complex at the telomere ends appears to be important in other organisms besides budding yeasts. Mutations in the mammalian telomere binding proteins TRF1 and TRF2 have been shown to result in varying degrees of telomere lengthening and chromosome fusions, respectively . In Schizosaccharomyces pombe , the telomere binding protein Taz1p has been shown to be important in telomere length control . Additionally, mutations in Taz1p that result in improper meiotic segregation, defects in telomere clustering, and low spore viability may reflect failure to form a functional cap . Understanding the role of capping in telomere function will likely be useful in understanding the roles of telomeres in cell viability and division control. | Study | biomedical | en | 0.999996 |
10209019 | Mitotic Xenopus egg extracts were isolated essentially as described by Hirano and Mitchison . In brief, the dejellied eggs were crushed in EB (80 mM β-glycerophosphate, pH 7.3, 15 mM MgCl 2 , 20 mM EGTA, and 1 mM DTT) supplemented with 10 μg/ml leupeptin and pepstatin, by centrifugation for 20 min at 20,000 g in an SW41 rotor ( Beckman Instruments ). The cytoplasmic fraction was collected and further fractionated by ultracentrifugation at 250,000 g for 2 h at 4°C by using the TLS-55 rotor ( Beckman Instruments ). The lipid layer was sucked very carefully under vacuum, the soluble fraction was removed and recentrifuged for an additional 30 min at 250,000 g in order to get rid of the residual membranes. The extract was aliquoted in 25-μl fractions and immediately frozen and stored at −80°C. Demembranated Xenopus sperm nuclei were prepared following the procedure of Smythe and Newport and stored at −80°C. Chromosome were assembled essentially as described by Hirano and Mitchison . Observations were made by an inverted microscope, using 60× phase-contrast objectives (NA = 0.6 or 1.3). Images were acquired through a CCD camera, and recorded on a VCR. When digitized, the resolution of images is 0.21 or 0.4 μm/pixel, depending on the objective used. Two micromanipulators were used to direct micropipettes and grab a chromosome by its ends. Micropipettes were formed to a final inner diameter of 1 μm using a puller (Sutter P-97). To have cylindrical pipette tips, their last 100-μm length was cut by a laboratory-made forge and fire polished. The deflection of micropipette tips was used to measure forces. A pipette acts like a normal spring, and the force F it applies is proportional to its tip deflection x (for small deflection compared with its length): F = kx , where k is the spring constant. The calibration of pipettes was done in two steps. First, a spring of known rigidity (0.84 × 10 −2 N/m) was used under the microscope to calibrate an intermediate pipette (rigidity constant 2.4 × 10 −3 N/m) which in turn calibrates micropipettes used for elasticity measurements (2–3 × 10 −4 N/m). For each pipette, the linearity of the spring was checked over 100 μm deflection, and the error was estimated to be <10%. Deflection of the micropipettes was measured by laboratory-made image correlation techniques and the minimum detectable displacement was ∼80 nm. A thin rod of section S and length L submitted to a force F along its axis would be elongated to L + Δ L . The strain was defined as ε = Δ L/L and was dimensionless. The Young modulus is the proportionality factor between the force per unit of area and the strain: F/S = Y ε. Y has the dimension of a pressure, or energy per unit of volume. The energy E needed to bend the thin rod of length L along a circle of radius R reads E = BL/R 2 . B has the dimension of an energy multiplied by a length. The resistance to bending of small object like polymers, actin filament, etc., is measured in units of the energy of thermal noise available in the bath, i.e., KT , where K is the Boltzmann constant and T the temperature (at room temperature 300 K, KT = 4.1 × 10 −21 J). Thermal noise induces random bending of the object on a scale called the persistence length, L p = B/KT . If the filament is shorter than L P , it will look like a straight rod (for example, L P is in the range of few millimeters for microtubules, thus microtubules of 10-μm length seem straight under the microscope). On the other hand, if the filament is much longer than L P , many random bends could be observed along its length. The measurement of these random bends is a powerful way of measuring the bending rigidity of a filament without any mechanical manipulation. Consider tangents at two points belonging to the filaments . If the points are close to each other (compared with L P ), their tangent will point to nearly the same direction, and the angle θ between them will be close to 0. On the other hand, if the distance between the two points is much higher than L P , there is no correlation between the tangents, and on average, <θ\> = π/2. One can show the following relation for the average angle between tangents at two points: <cos(θ( s ))\> = exp (− s/L p ), where s is the arc length between the two points. <cos(θ)\>, which is called the tangent autocorrelation function, means the average of cos(θ) over all points along the filament separated by an arc length s . If the filament is confined to two dimensions, <cos(θ)\> = exp (− s/ 2 L p ) . The principle of persistence length measurement is the same as described in Ott et al. for actin filament. Condensed chromosomes were spread between two coverslips and the reservoir of ∼4-μm thickness was sealed. Images of freely fluctuating chromosomes were recorded for few minutes on a tape recorder. A laboratory-developed computer program digitized the images, and a geometrical curve (1 pixel thick) describing the chromosome was obtained by classical skeleton finding algorithms . Each curve has been reparametrized in order to have constant arc length between consecutive points. The tangent autocorrelation function (see above) has been calculated for each curve and averaged over images of one fluctuating chromosome. The result has been averaged over 16 different chromosomes (∼5,000 images have been used for averaging). The process is summarized in Fig. 2 , a and b. After completion of the chromosome assembly, 5 μl of the solution was deposited in a small reservoir of 20 mm in diameter and 4 mm high, and diluted in 300 μl of EB. A chromosome was grabbed at both ends by two micropipettes. The displacement of one micropipette deformed the chromosome, which in turn applied force to the fixed micropipette. Deflection of the fixed micropipette (which is calibrated at the end of the experiment) measured directly the force applied to the chromosomes (see above). The simultaneous measurement of the length of the chromosome and the deflection of the fixed pipette allowed the determination of the Young modulus . Chromosomes were assembled by incubating demembranated Xenopus sperm in mitotic high speed extract isolated from Xenopus eggs. The kinetics of chromosome assembly is shown in Fig. 1 . As seen, sperm nuclei undergo a series of well-defined structural changes and after 3 h of incubation individual, fully condensed and well-separated chromosomes are formed. We have performed two independent measurements in order to determine the flexural rigidity and the elongation deformability of these chromosomes (for definitions, see Materials and Methods). The first measurement uses the random bends induced by thermal fluctuations. The second one is performed using two micropipettes, the first to deform the chromosome, and the second to measure the force applied to it. The Young modulus can be deduced when deformations are small. The high deformation regime gives additional critical information on the inner structure of chromosomes. The in vitro assembled mitotic chromosomes allow for precise measurement of various elastic responses, since they are condensed in cell-free extracts and are easy to manipulate. Their use is critical for the measurement of the flexural rigidity: as they are in a cell-free extract, their random bends are induced by thermal fluctuation. On the other hand, the in vivo chromosomes are constrained by the presence of the cytoskeleton. Moreover, during mitosis, the curvature fluctuations of in vivo chromosomes are due to the nonthermal length fluctuations of microtubules and cannot be assumed to be thermal. Applying force inside the cytoplasm to measure directly the bending modulus presents the same difficulties. All these considerations make the in vitro assembled mitotic chromosomes very suitable for elastic measurements. We will show below that we can reasonably assume the similarity between in vitro and in vivo mitotic chromosomes. The flexibility of an object is characterized by its bending modulus B . The energy needed to bend the object is proportional to B , and to the square of the induced curvature. We will show below that the measure of this material property shed light on the inner structure of chromosome. This information is also important for a better understanding of the processes taking place at anaphase: during anaphase, sister chromosomes are separated and pulled toward the poles. The force the cell needs to exert in order to pull the chromosome depends on the bending modulus of the chromosomes. If chromosomes can be easily bent, the force which resists their movement (due to the viscosity of the cytoplasm and the constraints imposed by the cytoskeleton network) will be small. On the other hand, if chromosomes are stiff, when pulled toward the poles, they will remain straight and perpendicular to the direction of motion. In this case, pulling them through the cytoskeleton network would need a much higher force. For elongated objects on the micron and submicron scale, the thermal noise randomly bends the object on a scale called the persistence length L p , which is proportional to B ( B = KTL p , where K is the Boltzmann constant and T is the temperature) (see Materials and Methods). Hence, the analysis of the curvature fluctuation spectrum is a straightforward way of measuring the persistence length. In brief, in vitro assembled chromosomes were observed under the microscope and their thermal fluctuation was video recorded . Video records of 16 freely fluctuating chromosomes (representing ∼5,000 images) were analyzed, and the tangent autocorrelation function was computed. This function shows a perfect exponential decay over approximately one order of magnitude and the deduced chromosome persistence length was found to be 2.7 ± 0.1 μm ( B = 1.2 × 10 −26 J · m) . Note that this represents only a few times the diameter of the chromosome (0.8 μm) and it is comparable to the persistence length of actin filaments (∼10 μm) which are 100 times thinner. The small value of L p (i.e., high chromosome flexibility) is likely necessary for the achievement of the anaphase. Chromosomes were visualized by phase-contrast or fluorescent microscopy with Hoechst 258 used for labeling. No difference has been observed due to the presence of the dye at concentrations up to 10 −6 M. An independent end to end distance measurement of 30 nonlabeled chromosomes gave similar result: L p = 3 ± 0.5 μm (data not shown). The extensibility of an object upon the action of a force is characterized by its Young modulus Y . The Young modulus depends on the underlying structure of a material. A high Young modulus reflects the fact that a high force is needed to elongate the object. The Young modulus of metals is in the range of 10 10 to 10 11 Pa, with those of gels lays in the 10 6 to 10 7 Pa interval. Microtubules and actin filaments have a Young modulus of 10 9 Pa. During mitosis, chromosomes are submitted to elongation stress due to microtubules and motors. A careful measurement of chromosome elongation at different times and the knowledge of their Young modulus should allow the determination of the force exerted on them at different stages of mitosis, a subject still submitted to debate after the article published by Nicklas . In this work, we have measured the chromosome Young modulus, and we will discuss below the relevance of this elastic constant for the chromosome underlying structure. For a thin rod of section S and length L submitted to a force F along its axis, the Young modulus is defined as: Y = F/S (Δ L/L ) −1 , where Δ L is the elongation induced by the force (see Materials and Methods). We determined the chromosome Young modulus from its force–extension curve by using the micropipette technique. A chromosome was suspended between two micropipettes, using a small amount of aspiration. The chromosome was then stretched by moving one of the micropipettes at a constant speed, while the length of the chromosome and the deflection of the other micropipette (used as a nanodynanometer) were simultaneously measured after recording the images on a video recorder . For a small deformation (Δ L/L < 1) and stretching rate (<1 μm/s), the curve of force versus deformation is reversible over many cycles. Fig. 4 shows a typical measurement for two different chromosomes. The Young modulus of chromosomes (measurements performed on 11 different chromosomes) is found to be in the 800–1,350 Pa range, with an average value of 1,100 Pa. Chromosomes can be elongated manifolds. For higher deformations, hysteresis is observed . The elasticity ceases to be linear and the behavior is similar to that observed for long biological polymers such as titin and tenascin . Successive deformation cycles, where the final extension is gradually elevated, induce also an irreversible transition in chromosome structure: after each cycle, the chromosome is softened and the next stretch necessitates a lower force in order to reach the same elongation. The maximum length to which chromosomes can be extended is variable: disruption of different chromosomes is observed for elongation between 12 and 100 times their original length. The force– extension curve of chromosomes that did not break after being stretched ∼15 times their original length show a plateau ∼5–10 nN . This plateau is associated with the partial unraveling of the chromosomes: chromosome parts exhibiting similar diameter as the nondeformed ones are found separated from each other and are connected by thin filaments. The thin filaments are not observable under the microscope, but their presence can be demonstrated upon moving the micropipette which induces displacement of the whole structure . Chromosomes were assembled from Xenopus sperm nuclei. Since these structures showed unusual elastic properties, we have been further interested to understand whether this behavior was specific to mitotic chromosomes only, or if it could be observed also on the “parental” structure, i.e., the sperm nucleus. In contrast to mitotic chromosomes, demembranated sperm nuclei appear as very rigid objects for both flexural and longitudinal stress. The Young modulus Y of sperm nuclei has been measured by the same micropipette technique as described above. The value of Y , measured on 11 different sperm nuclei, was found to be 200 ± 50 kPa. Note that this value is two orders of magnitude higher than those of mitotic chromosomes. The bending rigidity B of the sperm nucleus is too high to allow measurable thermal fluctuations. Thus, we used the micropipette directly to induce bending of sperms, and measured the force–curvature relation. The value of the bending rigidity obtained from three different samples was found to be 1 ± 0.5 × 10 −20 J · m, which corresponds to a persistence length of 2.5 m! This is six orders of magnitude higher than for mitotic chromosome. In this work we have studied the elastic behavior of in vitro assembled chromosomes in Xenopus egg extracts. Let us summarize the results obtained: we have measured a chromosome Young modulus of 1,100 Pa, and a persistence length of 2.7 μm. The relations between these values and the nonlinear high deformation behavior have profound consequences on the underlying chromosome structure and lead us to propose a model for the inner organization of mitotic chromosomes. Below, we will first discuss the fact that in vitro assembled chromosomes have similar substructures as their somatic counterparts. Next, we will show that our data strongly suggest the existence of thin rigid chromosome axes essential for mitotic chromosome organization. Finally, we will demonstrate that the nonlinear force–extension relationship observed for high deformations requires these rigid axes to be built similarly to an ensemble of highly elastic proteins. An important question to be addressed regarding the data presented is to what extent in vitro assembled chromosomes are related to the in vivo ones. Both types of chromosomes possess a similar overall shape and size as well as biochemical compositions . But their internal structures are not really known and the subject is still controversial . Determination of their material properties is well-adapted to answer this question. The Young modulus of chromosomes for small deformations has been measured by Nicklas for grasshopper anaphase chromosomes (500 Pa) and by Houchmandzadeh et al. for metaphase newt lung chromosomes (1,000 Pa). Moreover, the last authors showed that chromosomes can be stretched up to 100 times their initial length, and if the deformation is less than 10, their original length can be restored. Compared with these results, in vitro assembled Xenopus chromosomes are very similar: their Young modulus is within the same range as somatic ones (1,100 Pa), their original length can be restored for 5–10 times deformations, and they can be plastically deformed up to 100 times. All these similarities lead us to conclude that the internal structures of both assembled in vivo and Xenopus egg extract chromosomes are essentially identical. Let us summarize the results presented in the previous sections: the persistence length L p (which characterizes the resistance to bending) of chromosomes is 2.7 μm, while their Young's modulus Y (which is a measure of their resistance to stretching) is of the order of 1,000 Pa. No information about the underlying structure can be obtained from each value taken independently, but their relation has profound consequences. For a large class of material organizations, the relation between L p and Y obeys the following relation : 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{L}}_{p}={\mathit{CYr}}^{4}/{\mathit{KT}}\end{equation*}\end{document} where r is the radius of the object, C a numerical constant of order of unity (π/4 for a solid cylinder), T the absolute temperature, and K the Boltzmann factor. This relation is due to the fact that bending (with elastic response proportional to L p ) induces elongation/compression above/below a central neutral plane, with an elastic response that is proportional to Y . Eq. 1 would hold, for example, if the chromosome were formed of a crystalline polymer, if it were a gel-like material (the chromatin fiber cross-linked to itself by some molecular agent at random intervals), or, most importantly, if it were formed of a thinner fiber which folded helically to make the whole chromosome. Examples of biological objects obeying Eq. 1 are microtubules and actin filaments . In fact, Eq. 1 does not hold for the chromosome: as the radius of the chromosome is 0.4 μm, the right hand of Eq. 1 equals ∼5 mm, which is 2,000 times higher than the measured value of the persistence length. Roughly speaking, chromosomes appear to be hard when stretched, but very soft when bent. To explain our results, one has to imagine a model of chromosome organization in which the two apparent modes of elasticity (bending and stretching of the whole structure) are not related by Eq. 1 . A straightforward explanation is to suppose that the chromosome is formed of a thin rigid (and elastic) core, surrounded by a soft envelope. As the core is thin and the persistence length varies as the fourth power of the radius, the whole object can be bent easily, while its stretching implies large forces. As a commonplace example, this is similar to the case of electric wires, which can be bent easily because they are formed of many thin filaments of copper, but cannot be elongated. In such a model, chromosomes would possess a core with a Young's modulus higher than 10 6 Pa and a diameter <20 nm, while the Young's modulus of the surrounding soft envelope would be <1 Pa. In the limit case where the diameter of the core is 20 nm, the resistance to bending and stretching of the whole chromosome is ensured only by the thin axis and there is no contribution from the soft envelope. If, on the other hand, the diameter of the axis is much <20 nm, the resistance to bending of the thin axis becomes negligible: the persistence length of the whole chromosome is due only to the soft envelope, while its resistance to stretching is due only to the thin axis. In this latter case, bending and stretching of the whole chromosome involve independent elastic elements (see Appendix ) and each elastic element can be treated separately. Moreover, if we suppose that the diameter of the axis lies in the molecular range (and has negligible persistence length), one does not even need to assume that the core itself obeys classical elasticity, the only important attribute of the core being its resistance to elongation. This resistance, for the whole range of extension, can be computed using classical polymer theory, as for proteins such as titin and tenascin . Both of the above limit cases can consistently explain the relation between the measured values of the elastic constants of chromosomes. We will show below that deforming chromosomes by a large amount favors the second hypothesis. For simplicity in our calculations we have considered only one rigid axis. However, one can envisage several such axes distributed through the whole section of the chromosome, connected to each other by the soft envelope and constituting a columnar phase. From the measurement of L p and Y it is not possible to estimate the number and the space distribution of these axes. However, by using the same calculation, one can prove that the thickness of each axis should be <20 nm, and that their Young's modulus should be of the order of Y in / N , where N is the number of axes and Y in the Young's modulus of the inner structure for the one-axis case. Before going further, note that Eq. 1 holds for the sperm nucleus, the other very compact organization of DNA we have measured. Thus, sperm nuclei should have a homogeneous or helical substructure. The remodeling of the very condensed sperm nucleus in the Xenopus extract is associated with the removal of the protamine-like proteins and the uptake of numerous histone and nonhistone proteins . Our data show that these changes in protein composition result in dramatic changes in the underlying structure. How are the rigid axes built? As shown above , chromosomes can be stretched by many times their length; therefore, the axes store a large reservoir of length. Moreover, the chromosome force–extension curve shows a strong hysteresis and a gradual softening. Recently, similar force–extension behavior has been reported for a class of proteins that include titin and tenascin , and the presence of titin inside mitotic chromosomes has also been demonstrated . Native titin is a long (1 μm) polymer, formed of ∼250 Ig domains. Each domain, in its native form, has a length of ∼4 nm. Upon stretching, each domain can unfold and reach an overall length of ∼30 nm. When submitted to a gradually increasing force, the titin molecule first straightens from a coiled form, and when the force reaches a critical value, domains begin to unfold gradually. The elasticity of titin (and tenascin) is well-described by a modified worm-like chain model . The solid lines of Fig. 5 represent numerical resolution of several cycles of chromosome elongation, assuming 45 titin-like molecules per chromosome. The equation describing the force–elongation relation of these polymers is detailed in Kellermayer et al. and Rief et al. . Note that the persistence length of each titin molecule in this model is 2 nm. The total contribution of titin-like axes to the persistence length of the whole chromosome is <100 nm. Thus, the persistence length of the chromosome (3 μm) is mostly due to the chromatin matrix. The chromatin matrix itself does not contribute to the resistance to elongation. This model belongs to the limit case discussed above, where the resistance to bending and stretching of the whole chromosome is supposed to be due to independent elastic substructures. To explain the softening of the chromosome after a deformation cycle, we have assumed that, after each cycle, a fraction of unfolded domains fails to refold. Similar behavior has been observed for the elasticity of titin . As seen, the model is in impressive agreement with our experimental data and this implies that the rigid axes are built like titin molecules. Thus, proteins or protein complexes possessing titin-like elastic properties should be the main components of the chromosome axes. What are these proteins? At present we are not in a position to give a definite answer. Obviously, a good candidate may be titin itself, since it has been found to be associated with mitotic chromosomes . Other plausible candidates are the proteins from the SMC family . These proteins exist in Xenopus egg extract as high molecular complexes called condensins . Condensin immunodepleted extract is no longer able to assemble chromosomes, and thus condensins are essential for chromosome condensation and architecture . In addition, it was shown that bacterial SMC proteins have their coiled-coil domains organized around a hinge , which seems to be structurally flexible. Since the SMC proteins are very conservative, condesins could conceivably exhibit similar hinge structure. If this is really the case, the chromosome resistance to elongation will be determined by the elastic properties of the hinge. Recently, a model for chromosome condensation based on the “scissoring” action of the SMC proteins was proposed by Hirano . The presence of a single backbone of nonhistone proteins (a scaffold), responsible for the organization of metaphase chromosomes, has been postulated in the past . However, the existence of the scaffold structure in intact mitotic chromosomes has remained controversial . Indeed, the scaffold was observed only upon treatment of mitotic chromosomes with different detergents and salts and thus it was not clear whether the observed structure was not an artifact due to the precipitation of high molecular weight chromosomal proteins . For example, the first biochemically defined component of the scaffold was topo II, but depletion of topo II from in vitro assembled chromosomes did not change their elastic properties (data not shown). Additionally, within the most sophisticated version of this model, the scaffold and the chromatin loops form an ∼200-nm fiber , which further folds helically to assemble the mitotic chromosome. However, such a structure is not compatible with the measured chromosome elasticity: for this model, the diameter of the object (the outer diameter of the supposed helix) which ensures the elastic responses is of the order of the chromosome thickness. Hence, if the measured Young modulus is 1,000 Pa, the persistence length would be in the millimeter range, three orders of magnitude larger than the measured value. The same argument can be made for other model of chromosome structure , hierarchical helical folding , which cannot explain the apparent discrepancy between the measured values of chromosome elasticity. In our previous work on in vivo chromosomes , based on a limited amount of information, we had favored a model of chromosome organization based on helical folding of a thin filament. The diameter of the thin filament was supposed to be four times smaller than that of the mitotic chromosome. The argument was based on the huge extensibility of chromosomes, on the knowledge of their Young's modulus, and on geometrical considerations. Two critical data were missing at that time: the value of the persistence length of chromosomes and the force–elongation relation for high chromosome deformation, which are, as stressed above, very difficult to measure in vivo. These data have been obtained in this work, and as shown above, are not compatible with the model of helical organization. In conclusion, we have developed a novel approach for studying chromosome structure. Our experiments show that mitotic chromosomes exhibit specific elastic responses. Therefore, we propose a model for chromosome structure based on a single or several thin elastic axes surrounded by a soft envelope . The axes are proposed to consist of elastic titin-like molecules, while the envelope contains chromatin. The chromatin material is attached to the axes and is responsible for keeping the axes close together. The resistance of mitotic chromosomes to bending is ensured by the chromatin matrix, where the axes are responsible for the resistance of chromosome to elongation. The condensation state of the chromosome is determined by the interplay between the entropic forces exerted by the axes which tend to collapse them into a random coil and the excluded volume effects due to chromatin . Moreover, local decondensation of chromosomes can be achieved by a simple unfolding of axis protein domains. | Study | biomedical | en | 0.999997 |
10209020 | mAbs to the N-tail of H3 were prepared using, as an immunogen, a synthetic peptide of residues 1–17 in which Ser10 was phosphorylated and coupled to a keyhole limpet hemocyanin. The antibodies were purified from mouse ascites fluid by cycling over a protein A column. The mAb recognizing histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and the mAb recognizing both unphosphorylated and phosphorylated histone H3 N-tails (H3 Ab) were of the IgG2aκ and IgG1κ isotypes, respectively. Antibodies were stored at 3.7 mg/ml in 200 mM NaCl, 100 mM Hepes, pH 7.4, 7.7 mM NaN 3 , and 5 mg/ml BSA at 4°C. MCF-7 human breast carcinoma cells were cultured as monolayers . To obtain mitotic populations, 100 ng/ml nocodazole ( Sigma Chemical Co. ), from a 10,000-fold stock in DMSO, was added to growth medium for 18 h. The loosely attached mitotic cells were released into the medium, by tapping the culture flask, and collected. Mitotic populations contained between 60 and 90% mitotic cells, as determined by microscopy. Treatment with staurosporine ( Sigma Chemical Co. ) lasted for 15–30 min at 100 ng/ml, from a 1,000-fold stock in DMSO. Extracts from samples containing either 3 or 60% mitotic cells were prepared in 1 mM EGTA, 1 mM PMSF, and 4 μg/ml leupeptin. Protein content was determined by protein assay (Bio-Rad Laboratories). For ELISA, cell extracts containing 1–10 μg protein, or samples in PBS containing 0–0.5 μg BSA conjugated H3 peptide (residues 1–17), either phosphorylated or not at Ser10, were applied to 96-well plates (Nunc-Immuno) and blocked for 1 h in KB (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 1% BSA). The plates were incubated with 100 μl of H3 or H3P Ab, serially diluted in KB and HRP-conjugated goat anti–mouse IgG secondary antibody (Pierce) diluted 1:7,500 in KB for 1 h at ambient temperature. The plates were washed five times with 10 mM Tris-HCl, pH 7.4, 0.02% Tween 20. Antigen–antibody complexes were visualized by incubating with 120 mM Na 2 HPO 4 , 100 mM citric acid, pH 4.0, 0.5 mg/ml 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.01% hydrogen peroxide. Absorbance was measured at 405 nm with a Biotek plate reader. For Western blots, equal amounts of protein from cell extracts were separated using 15% SDS-PAGE. Proteins were transferred to nitrocellulose membranes by electroblot technique and membranes were blocked in PBS-T (PBS, 0.05% Tween 20) containing 5% nonfat dried milk, for 1 h at ambient temperature. The membranes were incubated for 1 h by shaking with antibodies diluted 1:10 6 in PBS-T and washed three times for 20 min each. The membranes were further incubated for 1 h with HRP-conjugated goat anti–mouse IgG secondary antibody ( Pierce Chemical Co. ) diluted 1:10,000 in PBS-T, washed three times for 10 min each, and washed once with PBS-T containing 1 M NaCl. Antibody binding was detected by enhanced chemiluminescence ( Pierce Chemical Co. ). Indirect immunofluorescence was performed as described previously , using the H3 Ab, H3P Ab, or H11-4 clone ( Boehringer Mannheim ) at a dilution of 1:400 in KB for 1 h at ambient temperature. Indirect immunofluorescence using mAb SPM8-2 was performed as in Delcros et al. . Cells were photographed on Kodak Tmax 400 film using a Zeiss axiophot microscope. Immunofluorescence quantitation was performed by scanning the negatives with a scanner ( Kodak professional plus RFS 2035 and measuring fluorescence intensity using image processing software (IPLab Gel 1.5e; Signal Analytics). To compensate for differences in photographic exposure between frames, the fluorescence intensity of mitotic cells was measured relative to that of adjacent interphase cells in the same frame. UV laser irradiation was performed using a Quanta-Ray pulsed Nd:YAG laser (model GCR14S; Spectra Physics) equipped with an HG-2 harmonic generator (Spectra Physics) and dichroic mirrors (DHS-2 Quanta-Ray dichroic harmonic separator) to give monochromatic 266-nm light with a beam diameter of 6.4 mm. Open 1.5-ml microfuge tubes, containing cell suspensions ready for irradiation, were placed horizontally in a 10-mm-diam hole drilled in a small Plexiglas™ sheet held in a Brinkmann micromanipulator. All experiments were performed using a single 5-ns pulse with an energy of 50 mJ measured with a power and energy meter (model AA30; Astral) equipped with a UV sensor (model AC25; Scientech) . Cycling and mitotic cells were collected and samples removed to determine the percentage of cells in mitosis. The cells were counted with a hemacytometer, washed twice with ice-cold PBS, and diluted in PBS in 1.5-ml microfuge tubes to give 5 × 10 6 cells an optical density of 5 OD 266 /ml. Equal cell samples were irradiated with a single 5-ns, 50-mJ pulse of 266 nm light. All of the following procedures were performed at 4°C unless stated otherwise. After irradiation, cells were lysed in RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), DNA was sheared by passing the lysate through a 21-gauge needle, and lysates were precleared for 30 min with 20 μl of a 50% slurry of protein A–agarose beads ( GIBCO BRL ). Samples of precleared lysates were separated by SDS-PAGE and stained with Coomassie blue to ensure that they contained the same amount of protein. Free H3 and cross-linked H3–DNA complexes were immunoprecipitated from precleared lysates by incubation with 3 μl of H3 Ab for 1 h, followed by incubation with 20 μl protein A–agarose beads for 1–2 h. Samples were centrifuged at 200 g and beads were washed once with RIPA, twice with RIPA containing 1 M NaCl, once with RIPA containing 0.25 M LiCl, and once with PBS. Immunoprecipitated H3– DNA complexes were digested by incubating the beads with 15 U micrococcal nuclease ( Pharmacia ) for 30 min at 37°C. The beads were washed twice with PBS, and DNA was end-labeled with [γ 32 P]ATP and 10 U T4 polynucleotide kinase ( Pharmacia ) for 30 min at 37°C. The beads were washed twice with RIPA and were incubated overnight with RIPA containing 1 M NaCl. Immunoprecipitated free H3 and phosphorus-32– labeled cross-linked H3–DNA complexes were eluted from the beads by boiling in SDS sample buffer containing 1 M urea . In some experiments, one-half of the sample was digested with SV8 protease ( Boehringer-Mannheim ) in 20 mM ammonium bicarbonate, pH 8.0, and two times the sample buffer for 4–12 h at ambient temperature. All samples were analyzed by 15% SDS-PAGE. Phosphorus-32 was quantified using a PhosphorImager (Molecular Dynamics, Inc.). Cycling or mitotic cells were collected, washed twice with PBS, suspended in serum-free DME ( GIBCO BRL ) containing 20 μCi [ 3 H]spermidine (NEN), and incubated with or without 100 ng/ml staurosporine for 30 min at 37°C in humidified 5%CO 2 . The cells were collected by centrifugation and washed three times with PBS. Compartmentalization of [ 3 H]spermidine was determined essentially as described in Mach et al. . Cells were lysed by pipetting for exactly 20 s in 500 μl 30 mM Hepes, 200 mM sucrose, 40 mM NaCl, 5 mM MgCl 2 , pH 7.6, and centrifuged for 20 s at 200 g . The cytoplasmic component contained in the supernatant was quickly removed and the procedure was repeated. The supernatant, designated the wash, was removed and the pellet, representing the nuclear compartment, was resuspended in 500 μl of the same buffer and vortexed vigorously. 3 H was detected by liquid scintillation counting. To study the involvement of the H3 N-tail in chromosome condensation, we first studied its phosphorylation at Ser10. We generated mAbs to a synthetic peptide (ARTKQTARK S TGGKAPR) representing residues 1–17 of H3 in which the Ser10 residue (underlined) was phosphorylated. In ELISA, an mAb designated H3P Ab recognized the peptide phosphorylated at Ser10 but not the unphosphorylated peptide , even when the antibody was used at high concentration. In Western blots, H3P Ab recognized a major band comigrating with H3 in mitotic cell lysates, where H3 was phosphorylated, and a very minor band at the same position in interphase cell lysates, where H3 was largely unphosphorylated . A minor band at 42 kD was also observed, but only in the mitotic cell lysates. The 42-kD band may represent H3 dimers arising from oxidation of H3 cysteine residues forming a disulfide bond, or it may be an unrelated protein with a similar phosphoepitope. A second mAb designated H3 Ab reacted equally well in ELISA with phosphorylated and unphosphorylated H3 peptide and with extracts from interphase and mitotic cells . In Western blots, it recognized H3 specifically and equally well in lysates from interphase or mitotic cells . To examine the phosphorylation of H3 at Ser10 during the different phases of the cell cycle we used H3P Ab in immunofluorescence microscopy in combination with the fluorescent DNA dye, bisbenzimide. Fig. 2 shows paired photographs of cycling MCF-7 cells with DNA fluorescence on the left (to define their cell cycle stage) and H3P Ab immunofluorescence on the right. In interphase cells (arrowheads), speckles of H3P Ab immunofluorescence were present in the nucleus that corresponded to regions of less intense DNA staining . Early in prophase, before chromosomes were clearly delineated , intense H3P Ab immunofluorescence appeared in the nucleus, associated with the condensing chromatin . H3P Ab immunofluorescence was also intense in metaphase , weaker in anaphase , but remained strongly associated with the chromosomes . In telophase cells, H3P Ab immunofluorescence was considerably reduced. In many cases it was absent entirely although the chromatin still appeared condensed , whereas in other cases, the periphery of each mass of chromatin showed no H3P Ab immunofluorescence but the center retained a patch of immunofluorescence (not shown). Throughout metaphase, anaphase, and telophase weak cytoplasmic immunofluorescence was also observed with the H3P Ab . These results indicate that in interphase a few discrete regions of nucleus that are low in DNA content, probably representing regions with less condensed chromatin, contain H3 phosphorylated at Ser10. A burst of phosphorylation on Ser10 takes place early in prophase at the onset of chromosome condensation, the phosphorylation is maintained at metaphase, reduced in anaphase, and disappears in telophase before or at the onset of chromosome decondensation. We performed a parallel immunofluorescence study using H3 Ab that recognizes both the phosphorylated and unphosphorylated H3 N-tail. As expected, cells in all stages of the cell cycle showed H3 Ab immunofluorescence in their nuclei . In interphase cells the pattern of H3 Ab immunofluorescence appeared the same as that of the DNA visualized with bisbenzimide . Unexpectedly, the intensity of H3 Ab immunofluorescence was distinctly higher in mitotic cells than in interphase cells . Early in prophase, increased nuclear H3 Ab immunofluorescence was observed that was associated with the condensing chromatin . H3 Ab immunofluorescence was intense in metaphase and somewhat weaker in anaphase , but remained associated with the chromosomes. In telophase cells, H3 Ab immunofluorescence was reduced further to levels approaching those observed in interphase nuclei . No cytoplasmic immunofluorescence was observed. The H3 Ab immunofluorescence from individual cells was measured by scanning densitometry. Immunofluorescence was significantly higher in prophase (2.4-fold), metaphase (3.3-fold), and anaphase (2.4-fold) cells than in interphase cells . Quantitation of the H3 Ab immunofluorescence signal using flow cytometry also showed a two- to threefold increase in mitotic cells over G2 cells (not shown). As an explanation for the increased H3 Ab immunofluorescence in mitosis, we considered the possibility that breakdown of the nuclear envelope in mitosis might generally increase the accessibility of nuclear targets to antibodies. However, a commercially available mAb (clone H11-4; Boehringer-Mannheim ) that reacts with most histones (H1 > H3 = H2A = H2B ≫ H4) showed equal staining of interphase and mitotic cells under identical experimental conditions and quantitation of the fluorescence signal by scanning densitometry showed no significant difference between interphase and mitotic cells . This indicates that antibodies can reach nuclear targets as readily in interphase as in mitotic cells. The increased binding of the H3 Ab to mitotic chromosomes could not be due to preferential binding to phosphorylated H3 or binding to additional chromosomal proteins at mitosis since Western blots showed that the H3 Ab recognized only a single band with the molecular mass of H3 in whole cell lysates from interphase or mitotic cells . Nor could the weaker binding of the H3 Ab in interphase nuclei be due to masking of the H3 Ab epitope by a posttranslational modification of the H3 tail, such as methylation or acetylation, because the H3 Ab reacted equally well with H3 from interphase or mitotic cells in Western blots and ELISA . These results show that the accessibility of the H3 N-tail to the H3 Ab increases when cells enter mitosis and is temporally correlated with the phosphorylation of H3 at Ser10. We considered the possibility that increased accessibility of the H3 N-tail to H3 Ab as chromatin changes from its decondensed interphase state to its condensed mitotic state, might occur because of a decrease in binding of the H3 N-tail to DNA. To examine the state of binding of the H3 N-tail to DNA in interphase and mitotic chromatin, we performed in vivo UV laser cross-linking using a single short (5 ns) pulse of irradiation. We developed this approach because of concerns that experiments carried out in vitro with isolated interphase nuclei and metaphase chromosomes do not reflect the in vivo situation, and because current procedures for cross-linking or footprinting in living cells require modification of DNA structure or integrity and long treatments that might disrupt interactions between the H3 N-tail and DNA. Stefanovsky et al. showed that irradiation of isolated nuclei or chromatin using short pulses of UV laser irradiation causes covalent cross-links between histones and DNA, via the histone tails exclusively. Irradiation with nano- or pico-second lasers causes the cross-links to form in <1 μs , well below the 100-μs timescale for conformational transitions of macromolecules . Therefore, we used a laser to irradiate living cells with a single 50-mJ, 5-ns pulse of 266 nm UV light. We lysed the cells, immunoprecipitated H3 under stringent conditions, enzymatically trimmed the DNA cross-linked to H3, end-labeled it with phosphorus-32, released the complexes from the beads used in immunoprecipitation, and analyzed them using SDS-PAGE and quantitation with a PhosphorImager. The radiolabeled band seen in SDS-PAGE represents H3 cross-linked to DNA. As outlined in Materials and Methods, irradiation was performed on samples containing the same number of cells at the same absorbance at 266 nm and immunoprecipitations were performed on samples containing the same amount of protein. H3 Ab immunoprecipitates from irradiated cycling cells contained a major radiolabeled band corresponding to H3 that was not observed in unirradiated controls . Microscopy of a sample of the original cell population showed it to contain 3% mitotic cells. The H3 Ab immunoprecipitates from an irradiated mitotic population (85% mitotic cells) also contained a radiolabeled band corresponding to H3 but its intensity was reduced to 56% of the interphase band. This result was observed in five experiments, with reductions ranging from 46% to ∼100% (not shown). Therefore, H3 is cross-linked to DNA to a larger extent in interphase cells than in mitotic cells. These results indicate that when cells enter mitosis a phosphorylation-dependent mechanism weakens the association of the H3 N-tail with DNA and that this weakening is important for chromosome condensation. To confirm cross-linking had occurred via the H3 N-tail and not via the histone fold domain, cross-linked H3– DNA complexes immunoprecipitated from interphase cells and labeled with phosphorus-32 were digested with SV8 protease. SV8 protease cleaves H3 into one 6-kD fragment corresponding to the N-tail (residues 1–50), and several fragments too small to visualize on 15% SDS-PAGE. The band corresponding to intact H3 disappeared after SV8 protease digestion and a band of similar intensity appeared at a position corresponding to the 6-kD cleavage fragment . This result demonstrates that cross-linking of H3 to DNA occurred via the N-tail. A radioactive band below the H3 N-tail band in the SV8-digested sample was also observed in unirradiated samples and in samples immunoprecipitated with unrelated antibodies. Therefore, it does not represent DNA cross-linked to a histone H3 proteolytic fragment, but rather it is a proteolytic fragment of immunoglobulins that became phosphorylated during incubation with [γ- 32 P]ATP. To examine whether the rearrangement of the H3 N-tail with DNA at mitosis resulted from Ser10 phosphorylation, we treated mitotic cells with the protein kinase inhibitor, staurosporine, which causes rapid dephosphorylation of histone H1 and of H3 Ser10 , and performed immunofluorescence and cross-linking experiments. Cells arrested in mitosis with nocodazole were treated with or without 100 ng/ml staurosporine for 20 min and prepared for fluorescence microscopy using H3 or H3P Abs and bisbenzimide. Some cells were prepared as chromosome spreads so that the state of condensation could be evaluated more clearly. In all cells, staurosporine caused rapid chromosome decondensation, but the extent varied between individual cells. This is best shown in the chromosome spreads . In most cases staurosporine caused the discrete highly condensed mitotic chromosomes to decondense into a mass of chromatin . In other cases chromosomes, although decondensed, could still be discerned . Immunofluorescence microscopy showed that after treatment with staurosporine, not only were mitotic chromosomes decondensed into fuzzy masses of chromatin , but the intense H3 Ab immunofluorescence associated with mitotic chromosomes of untreated cells was reduced significantly . Each frame in Fig. 6 was exposed differently to optimize detail, but in each case the intensity of H3 Ab immunofluorescence in mitotic cells can be compared with an interphase cell in the same preparation. In untreated cells , the H3 Ab immunofluorescence intensity of the two mitotic cells is sufficiently high that at an appropriate exposure no immunofluorescence was seen in the interphase cell. After treatment with staurosporine, the partially decondensed chromosomes seen in the cell in Fig. 6 E (arrow) show much less H3 Ab immunofluorescence than untreated cells , even though a longer exposure was used so that H3 Ab immunofluorescence may be detected in the adjacent interphase cells . In some cells, staurosporine treatment resulted in more extensive decondensation of chromosomes and the H3 Ab immunofluorescence was correspondingly weaker , approaching that of adjacent interphase cells. Quantitation using scanning densitometry showed that the fluorescence signal decreased from 3.26 ± 1.35 ( n = 6) to 1.15 ± 0.36 ( n = 6) during a 20-min staurosporine treatment. Similar experiments performed using the H3P Ab confirmed that staurosporine caused H3 dephosphorylation; a cell with partially decondensed chromosomes no longer showed chromosomal H3P Ab immunofluorescence , but showed cytoplasmic staining at an exposure long enough to show the speckled H3P Ab immunofluorescence of interphase nuclei . We also examined the effects of staurosporine treatment on the association of the H3 N-tail with DNA. Samples containing equal numbers of cells arrested in mitosis with nocodazole were either treated or not treated with staurosporine for 20 min and irradiated. Staurosporine treatment led to an increase in the amount of radiolabeled H3 from 56% to 84% , indicating that binding of the H3 N-tail to DNA increased during staurosporine-induced chromosome decondensation. These results show that experimental induction of chromosome decondensation and H3 dephosphorylation leads to a decrease in the accessibility of the H3 N-tail to antibodies and an increase in its association with DNA. The results strengthen our earlier correlation between the conformation of H3 N-tail and chromosome condensation and Ser10 phosphorylation. The results also show that a staurosporine-sensitive kinase regulates the accessibility of the H3 N-tail to antibodies and its binding to DNA. Close packing of nucleosomes is expected to require neutralization of some DNA negative charge to reduce repulsion between DNA segments . It was presumed that this is accomplished in part by the positively charged H3 N-tail associating with linker DNA . According to this view, the dissociation of the N-tail from DNA during chromosome condensation that we observe would be expected to hinder chromatin compaction, unless it served to facilitate the binding of other positively charged molecules or is a consequence of binding of other positively charged molecules to DNA. The most abundant cellular polycationic molecules are the polyamines, which can bind to DNA and associate with chromosomes at mitosis . To determine the dependence of this association on protein phosphorylation, interphase cells, mitotic cells, and mitotic cells treated with staurosporine were incubated with [ 3 H]spermidine, which accumulates rapidly in cells. After brief fixation, cytoplasmic and nuclear compartments were separated quickly and the association of [ 3 H]spermidine with these two compartments was determined . In all samples, most [ 3 H]spermidine was in the cytoplasmic compartment. However, a larger proportion was in the cytoplasmic fraction of interphase cells than of mitotic cells. Conversely, the nuclear compartment of mitotic cells contained more [ 3 H]spermidine (33%) than that of interphase cells (15%). Staurosporine treatment of mitotic cells caused a decrease in [ 3 H]spermidine in the nuclear compartment to 21% accompanied by an increase in the cytoplasmic compartment to 65%. Therefore, in mitotic cells more spermidine is associated with the nuclear compartment than in interphase cells but treatment with staurosporine reduced this association. We were unable to use the UV laser cross-linking procedure to examine polyamine binding to DNA in vivo because polyamines do not form covalent cross-links at 266 nm (Sauvé, D.M., unpublished results). Further evidence for the sensitivity of chromosomal association of polyamines to staurosporine was obtained by indirect immunofluorescence with mAb SPM8-2 that recognizes spermine and spermidine . Polyamines are small and present in compartments unreachable by antibodies. To allow antibody access, cells rapidly fixed in formaldehyde were digested with DNaseI and RNaseA before incubation with antibody , although this treatment considerably reduces the quality of the preparations. In mitotic cells, polyamine immunofluorescence was more intense in the chromosomes than in the cytoplasm . After treatment with staurosporine for 15 min this difference was lost, leading to uniform immunofluorescence throughout the cell , indicating a redistribution of polyamines from the chromosomes to the cytoplasm and supporting the results obtained with [ 3 H]spermidine. Our H3P Ab immunofluorescence studies showed that Ser10 phosphorylation starts early in prophase, at the onset of chromosome condensation. The seemingly all or none appearance of Ser10 phosphorylation indicates that it occurs over a very short time period, attaining a maximum before the chromosomes become fully condensed at metaphase. In contrast to phosphorylation, dephosphorylation of Ser10 takes place over a longer time period because anaphase and telophase cells with intermediate levels of phosphorylation were often observed. Dephosphorylation precedes chromosome decondensation since telophase cells with condensed chromosomes showing little or no H3P Ab immunofluorescence were observed. The pattern of loss of H3P Ab immunofluorescence in telophase cells indicated that Ser10 dephosphorylation initiates at the periphery of the chromosome mass, suggesting that the dephosphorylation signal originates in the cytoplasm rather than within the chromosomes. Weak speckles of H3P Ab immunofluorescence were also observed in the nuclei of all interphase cells. In contrast with H3P Ab immunofluorescence in mitotic cells that is associated with all chromatin, these speckles were restricted to regions of less condensed chromatin. Western blots of interphase cell extracts showed a single minor band at the position of H3, implying that a small proportion of H3 is phosphorylated at Ser10 in interphase. This result is compatible with the observation that mitogen stimulation of quiescent interphase cells causes the rapid phosphorylation of a small fraction of H3 . This H3 fraction is very sensitive to acetylation , implying that it is associated with new gene expression that requires the opening up of chromatin. Our observations generally support the pioneering biochemical studies that first established a temporal link between H3 phosphorylation and chromosome condensation in mammalian cells and recent studies using a rabbit antiserum that recognizes phosphorylated H3 . Gurley et al. suggested that Ser10 phosphorylation began at the time of chromosome formation in prophase, reached a maximum in metaphase, diminished in anaphase, and was absent in telophase. However, we place the onset of Ser10 phosphorylation earlier, at the very beginning of prophase, when chromatin is beginning to condense but chromosomes are not clearly delineated. Hendzel et al. and Van Hooser et al. reported Ser10 phosphorylation to begin even earlier, in G2 and to initiate in pericentromeric heterochromatin. In our study, all cells showing increased H3P Ab immunofluorescence also showed morphological evidence of being in mitosis, suggesting that H3 phosphorylation takes place at the onset of mitosis and not in G2. We did not attempt to delineate precisely whether H3 phosphorylation initiates at specific positions within chromosomes. Our results are compatible with Ser10 phosphorylation playing a causative role in the initial stages of chromosome condensation, which involve compaction of the chromatin fiber, since phosphorylation is strong in early prophase, but not in the later stages of condensation, which involve coiling of the chromatids , since H3 phosphorylation peaks before maximal chromosome condensation and is absent in the condensed chromosomes of telophase cells. Similarly, Van Hooser et al. observed that hypotonic treatment of mitotic cells can cause H3 dephosphorylation without accompanying chromosome decondensation and suggested that H3 phosphorylation is not required for maintaining high levels of chromosome condensation. Whereas the H3P Ab recognized only a small subset of interphase H3 molecules, the H3 Ab showed staining of all interphase chromatin in a pattern indistinguishable from that obtained with the DNA dye, bisbenzimide. Unexpectedly, the accessibility of the H3 N-tail to the H3 Ab changed during mitosis: it increased sharply at prophase, was high at metaphase, decreased at anaphase, and decreased further to approach interphase levels at telophase. This pattern closely resembled that seen with the H3P Ab, suggesting that Ser10 phosphorylation and increased accessibility of the H3 N-tail are closely associated during mitosis. Our UV laser cross-linking experiments provide evidence that the H3 N-tails are indeed bound to DNA in vivo. They showed that the H3 N-tail is associated with DNA during interphase and that binding of the N-tail to DNA is decreased in cells arrested in metaphase by nocodazole. The most straightforward interpretation of our observation that the H3 N-tail becomes more accessible to the H3 Ab during mitosis, is that this occurs as a result of its dissociation from DNA. The results have several important implications. They are incompatible with compaction being driven by increased association of the H3 N-tail with DNA, but rather they provide evidence that compaction may be driven by dissociation of the tails from DNA. Dissociation could free the tails to bind proteins required for chromosome condensation, such as topoisomerase II or the SMC class , or allow access of the vacated DNA to factors required for condensation, as discussed below. Our observations also bear importantly on our understanding of the structure of the compact chromatin fiber. According to a widely accepted model , the compact fiber is a solenoid with about six nucleosomes per helical turn in which the nucleosomes are stacked with their long axis parallel to the fiber, with the DNA entry and exit sites and linker DNA inside the fiber. In this model, the H3 N-tails extend into the interior of the fiber, in a central hole too small to allow access to large antibody–secondary antibody complexes, an arrangement incompatible with our immunofluorescence observations. Previous biochemical observations asserting that linker DNA is more accessible to nuclease than intranucleosomal sites in condensed chromatin, and that histone H1 is the first histone degraded by trypsin are also incompatible with this model. Our results are more compatible with models where the H3 N-tail is not expected to preferentially face the inside of the fiber. These include the coiled linker model, in which linker DNA is on the outside of the fiber , different zigzag ribbon models containing two parallel rows of nucleosomes , and results from scanning force microscopy and cryoelectron microscopy showing an irregular pattern of folding, possibly a three-dimensional zigzag without face to face aggregation of the nucleosomes . Our results are also compatible with recent data obtained from X-irradiation of living cells that suggested a zigzag model of the chromatin fiber . Regardless of the organization of DNA and histones within the chromatin fiber, an essential constraint on the formation of compact chromatin is probably the neutralization of some of the negative charge of the linker DNA, to reduce repulsion between linker DNA segments . Dissociation of the H3 N-tail from DNA would achieve the opposite effect. Therefore, DNA charge neutralization during chromosome condensation must be accomplished by a different mechanism. Polyamines can displace histone N-tails in nucleosome core particles at concentrations >0.7 spermidine molecules per DNA turn and can cause chromatin compaction in vitro, with maximal compaction at about 1.5–2 polyamines per DNA turn . Human cells in culture typically contain 0.6–3 × 10 9 molecules of spermidine and spermine and 5–10 pg DNA, corresponding to 0.6–1.2 × 10 9 DNA turns. Therefore, the cellular concentration of polyamines appears sufficient to play a role in chromatin compaction in vivo. We examined whether polyamines play a role in mitotic chromosome condensation in vivo by labeling cells with [ 3 H]spermidine, rapidly separating the nuclear and cytoplasmic compartments, and performing immunofluorescence with an mAb to spermine and spermidine. We found that there was an increased association of [ 3 H]spermidine with the nuclear fraction in mitotic cells compared with interphase cells. In addition, polyamine immunofluorescence was found to associate preferentially with chromosomes. These findings agree with early fractionation studies showing high spermidine and spermine concentrations in mitotic chromosomes , and with immunocytochemical studies using a polyclonal antiserum to spermine and spermidine showing that polyamines are almost entirely cytoplasmic during interphase but associate with chromosomes in mitosis . Interestingly, Hougaard et al. observed that the association of spermine and spermidine with chromosomes occurred only in prophase, metaphase, and early anaphase, when we find that H3 is phosphorylated and more accessible to H3 Ab binding, and not in telophase, when H3 is dephosphorylated and becomes less accessible to H3 Ab binding. Treatment of mitotic cells with staurosporine concurrently caused the following: dephosphorylation of H3 Ser10, decondensation of chromosomes, decreased availability of the H3 N-tail to antibody binding, increased binding of H3 N-tail to DNA, and decreased association of polyamines with DNA. These events are interconnected and controlled by a staurosporine-sensitive protein kinase. We do not know the nature of the protein kinase(s) or its relevant substrates but it is tempting to speculate that the substrate may be H3 Ser10 itself. This is suggested by the temporal link between Ser10 phosphorylation and the onset of chromosome condensation. Phosphorylation of H3 Ser10 at prophase could weaken the interaction of the N-tail with DNA to allow binding of polyamines to DNA to drive chromosome compaction. Alternatively, the staurosporine-sensitive kinase(s) may control phosphorylation of other proteins, including histone H1, that also participate in the control of chromosome condensation. Currently, we are seeking this kinase. The observation that staurosporine treatment had no effect on the interphase speckles of H3P Ab immunofluorescence suggests that the kinase responsible for interphase H3 phosphorylation is not the mitotic H3 kinase. The results presented in this study show that a structural rearrangement of the H3 N-tail accompanies Ser10 phosphorylation at mitosis and are consistent with the following model. During interphase, the H3 N-tail is bound to DNA and participates in chromatin compaction, in concert with other histone tails and factors. The tails of H3 and other histones can be modified extensively during interphase by acetylation, methylation, ubiquitination, and phosphorylation. These modifications may serve to fine tune the degree of chromatin compaction to allow the regulated access to DNA that is required for diverse transactions such as transcription, replication, and repair. In calf thymus chromatin, 50% of the DNA charge is neutralized by histones . This may be sufficient for regulated chromatin compaction during interphase, but not for the extensive compaction required in mitosis. This function may be carried out by a distinct set of polycationic molecules more suited for bulk compaction, such as polyamines. Phosphorylation of the H3 N-tail at mitosis weakens N-tail–DNA binding and favors DNA–polyamine binding. Polyamines neutralize the negative charge of DNA more extensively, thus minimizing repulsion between nucleosomes and allowing formation of the highly compacted mitotic chromosome. Displacement of the H3 N-tail from DNA may also promote its association with other histones in adjacent nucleosomes or with nonhistone proteins to further stabilize condensed chromatin. Dephosphorylation of H3 Ser10 in anaphase and telophase would increase the affinity of the H3 N-tail to DNA and favor displacement of the polyamines, promoting chromatin decondensation. Finally, we saw no evidence of increased accessibility of the H3 N-tail in condensed heterochromatin compared with decondensed euchromatin in interphase nuclei. This implies that condensed mitotic chromosomes may be a form of compaction distinct from that of interphase heterochromatin in both structure and regulation. | Study | biomedical | en | 0.999996 |
10209021 | Human NUP98 cDNA was obtained as described . A full-length mouse RAE1 cDNA was generated from two partial cDNA clones, 473342 and 465642, obtained from the I.M.A.G.E. consortium. HA1-NUP98, mouse HA1-RAE1, and mutants thereof were generated by PCR using plaque-forming unit DNA polymerase (Stratagene Inc.), and cloned in pUHD10S for expression in HtTA cells . All constructs were verified by DNA sequence analysis. HA1-RAE1 cDNA was cloned into vector pSP73 ( Promega Corp. ) for in vitro translation purposes. [ 35 S]-methionine–labeled HA1-RAE1 protein was produced using the TNT-coupled rabbit reticulocyte lysate system ( Promega Corp. ) as indicated by the manufacturer. To isolate pure populations of transiently transfected HtTA and baby hamster kidney (BHK)grβ cells, HA1-NUP98(150–224) was cloned into the EcoRI–XhoI sites of vector MSCV-IRES-GFP . HtTA-1 and BHKgrβ cells were grown in DMEM containing 10% FBS. Cells were transfected with Superfect transfection reagent (QIAGEN Inc.) according to the manufacturer's instructions. Metabolic labeling of HtTA cells was as described . For indirect immunofluorescence studies, HtTA cells were transiently transfected in 24-well dishes, seeded on microscope slides 6-h after transfection, and stained 16–18 h later. To study the effect of the RNA–polymerase II inhibition on the RAE1 distribution in HtTA cells, the culture medium was supplemented with 0.04 or 5.0 μg/ml actinomycin D (AMD; Boehringer Mannheim Biochemicals ), or 50 μg/ml DRB (Fluka Chemical Corp.). To obtain pure populations of transiently transfected HtTA or BHKgrβ cells, green fluorescent protein (GFP)–positive cells were isolated by fluorescence-activated cell sorting . Electron microscopy was as previously described . To generate RAE1-specific antibodies, we cloned cDNA sequences encoding mouse RAE1 amino acids 188–347 into pQE30 (QIAGEN Inc.). HIS-tagged recombinant mouse RAE1 protein was produced in Escherichia coli DH12S cells, purified with Ni-NTA (nitrilotriacetic acid) agarose beads (QIAGEN Inc.) according to the manufacturer's instructions, and injected into rabbits. NUP98- and CRM1-specific antisera were generated as described . Antibodies were affinity purified using recombinant antigen bound to ProBlott ( Perkin-Elmer Corp. ) as previously reported . Indirect immunofluorescence, coimmunoprecipitations, and Western blot analyses were carried out as previously described in detail . A cDNA fragment encoding NUP98(150–224) was cloned in pQE31 and pGEX-5X-1 for expression of HIS- and glutathione- S -transferase (GST)– tagged recombinant NUP98(150–224). Recombinant protein expression in E . coli strain DH12S was induced by addition of 1 mM IPTG (isopropyl-β- d -thigalactopyranoside), followed by incubation at 25°C for 4–5 h. Harvested bacteria were suspended in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 2 mM PMSF, 1 mM leupeptin, 2 mM aprotinin, and 1 mM pepstatin. After 30 min of lysozyme treatment at 4°C, bacteria were lysed by sonication. HIS-NUP98(150–224) and GST-NUP98(150–224) were then purified from bacterial lysates using Ni-NTA agarose and glutathione beads (Sepharose 4B; Pharmacia LKB Biotechnology Inc. ), respectively (according to standard procedures), and used in pull-down assays. A cDNA fragment encoding RAE1(1–368) was cloned in pGEX-5X-1 to express GST-RAE1 in E . coli . Bacterial pellets were suspended in PBS with 2 mM PMSF, 1 mM leupeptin, 2 mM aprotinin, and 1 mM pepstatin. Lysis was performed by sonification and GST-RAE1 was purified with glutathione beads using standard procedures. Purified RAE1 was separated from the GST-affinity tag using Factor Xa protease ( Pharmacia LKB Biotechnology Inc. ). Glutathione beads with 100 ng purified GST-NUP98(150–224) were washed three times with binding buffer (20 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl 2 , 0.1% Tween-20, 20% glycerol, 0.01% bovine serum albumin, 1 mM dithiothreitol, 1 mM PMSF, 1 mM leupeptin, 2 mM aprotinin and 1 mM pepstatin), preblocked for 10 min with rabbit serum, washed with binding buffer, and resuspended in 60 μl binding buffer. Then, 10 μl in vitro–transcribed and –translated [ 35 S]-methionine–labeled HA-RAE1 was added to the beads and the mixture was incubated at 4°C for 1 h (vortexed every 5 min). Beads were washed six times with binding buffer and boiled in 15 μl SDS sample buffer. Samples were analyzed by SDS-PAGE (10% polyacrylamide), followed by autoradiography. Pull-down assays with Ni-NTA agarose aliquots containing 100 ng of HIS-NUP98(150–224) were performed in the same way. For chemical cross-linking, pellets were resuspended in 10 μl PBS with 1 mM disuccinimidyl suberate (DSS; Pierce) after the last wash in our pull-down protocol. Cross-linking was at 4°C for 20 min. Finally, 10 μl SDS sample buffer was added and boiled samples were analyzed by SDS-PAGE (9% polyacrylamide) followed by autoradiography. Trypsin digestion of in vitro–translated or recombinant RAE1 was as previously described . The reaction was stopped with 2 μl of 100 mM benzamidine. For enzymatic RNA degradation, 10 μl in vitro– transcribed and –translated [ 35 S]-methionine–labeled HA1-RAE1 was incubated with either 0.1 unit Micrococcal nuclease ( Sigma Chemical Co. ) or 10 μg RNase A. Both incubations were at 37°C for 30 min in the presence of 1 mM PMSF, 1 mM leupeptin, 2 mM aprotinin, and 1 mM pepstatin. HtTA or BHKgrβ cells were stained for specific proteins and poly(A) + RNA by use of the following combined immunostaining/in situ hybridization procedure. At 6 h after transfection, HtTA cells were seeded on microscope slides, and ∼16 h later they were fixed in PBS/3% formaldehyde for 15 min at 4°C. After five washes in PBS, cells were permeabilized in PBS/0.5% Triton X-100 for 5 min at 4°C. They were then washed in PBS (5×) and incubated with either 12CA5 antibodies (9 μg/ml) alone or 12CA5 and anti–RAE1 antibodies in PBS/2% BSA/0.2% Triton X-100/ 200 U/ml RNasin for 30 min at room temperature (RT). Cells were then washed in PBS (5×) and incubated with fluorochrome-coupled secondary antibodies for 30 min at RT: RAE1 antibodies were detected with Texas red–conjugated goat anti–rabbit antibodies (5 μg/ml), and 12CA5 mouse monoclonal antibodies were detected with R-phycoerythrin–conjugated goat anti–mouse antibodies (Caltag Laboratories). Cells were washed in PBS (5×), and then fixed in PBS/3% formaldehyde for 5 min at RT. After five rinses in PBS, the cells were equilibrated in 2× SSC for the in situ hybridization. Hybridization was performed by using an FITC-coupled oligo-(dT) 50 mer probe as detailed by Amberg et al. . At the end of the procedure, the cells were mounted on the slides in Vectashield (Vector Laboratories, Inc.) and analyzed by using laser scanning confocal microscopy. The above procedure is very well suited for detection of alterations in nuclear poly(A) + RNA levels. However, due to the relatively short fixation time of 15 min, a fraction of the cytoplasmic poly(A) + RNA pool is lost. Therefore, if we wanted to study the cytoplasmic poly(A) + RNA levels in detail, we extended the 3% formaldehyde fixation step by 15 min and fixed at RT. [ 35 S]-methionine–labeled protein for microinjection into Xenopus oocytes was synthesized in a rabbit reticulocyte lysate as indicated by the manufacturer ( Promega Corp. ). Templates were pT7-CBP80 , pSP73-HA1-RAE1 (mouse), and pT7-GST-NES, encoding a GST fusion with the HIV-1 Rev nuclear export signal . E . coli –expressed Rna1p was coinjected at a concentration of 80 μM in the injection mixture as described . Microinjections, incubations, and protein extraction and analysis were performed as described . Nuclear export of RAE1 was quantified by measuring the nuclear and cytoplasmic fractions of RAE1 with a phosphoimager (we corrected for leakage of RAE1 into the cytoplasm by quantifying the percentage of cytoplasmic CBP80). We overexpressed an HA1-tagged NUP98 cDNA in HtTA cells and immunoprecipitated the cell lysates with 12CA5 monoclonal antibody against the tag. A protein of ∼40 kD specifically coprecipitated with HA1-NUP98 . We wanted to test whether this interacting protein was the human homologue of the S . cerevisiae Gle2p because of (a) the molecular weight similarity and (b) the presence of a motif within NUP98 that is similar to the Gle2p-binding sequence of Nup116p . We obtained a murine cDNA clone with similarity to yeast gle2/rae1 cDNA (designated RAE1 ) and generated polyclonal antisera in rabbits against the carboxy-terminal half of RAE1. We overexpressed HA1-NUP98 in HtTA cells, immunoprecipitated the cell lysates with 12CA5 monoclonal antibody against the HA1 tag, and performed a Western blot analysis using affinity-purified antibodies raised against mouse RAE1. As shown in Fig. 1 C, the RAE1 antibodies recognized the HA1-NUP98 coprecipitating protein, demonstrating that NUP98 indeed interacts with the human homologue of the yeast Gle2p. To ensure that the interaction of NUP98 with RAE1 was not an overexpression artifact, we precipitated NUP98 from nontransfected HtTA cells with affinity-purified NUP98 antibodies and determined whether RAE1 was coisolated by Western blot analysis. RAE1 indeed coprecipitated with NUP98 from HtTA cells . When affinity-purified RAE1 antibodies were used in the immunoprecipitation step, NUP98 was coisolated with RAE1 , thereby confirming that NUP98 and RAE1 are in a complex in human cells. It should be emphasized that our data do not rule out the possibility that the NUP98–RAE1 complex is part of a larger protein assembly. To test the role of the GLEBS-like motif of NUP98 in RAE1 binding, an HA1-NUP98 mutant lacking amino acids 192–221 was generated . This mutant, designated as HA1-NUP98Δ(192–221), failed to coimmunoprecipitate RAE1 , although, like full-length NUP98, it localized at the NE (data not shown). Hence, the GLEBS-like motif of NUP98 was necessary for binding RAE1. We then asked whether this region of NUP98 was sufficient for RAE1 binding. We expressed amino acids 150–224 of NUP98 as an HA1-tagged fusion protein in HtTA cells and performed a co-IP Western analysis. As shown in Fig. 1 G (lane 2), NUP98(150–224) indeed coimmunoprecipitated RAE1. Additional mutagenesis studies revealed that the actual NUP98 interaction motif is located within residues 181–224 . Computer analysis (using the GCG program PEPTIDESTRUCTURE) identified a potential alpha-helical region from amino acids 187–212. Three helix-breaking proline mutations introduced in this region abrogated the NUP98(181–224) interaction with RAE1 , further confirming that the NUP98(181–224) segment contains the GLEBS-like motif. To further characterize the RAE1–NUP98 interaction, we produced RAE1 and the GLEBS-like motif in vitro, and analyzed whether they bind directly or indirectly via an adaptor protein or a molecule of mRNA. Presumably, members of the WD-repeat superfamily all form compact globular propeller structures that are resistant to proteolysis . It has been shown previously that most WD-repeat proteins fold into their native globular structure when synthesized in vitro in a rabbit reticulocyte lysate system, but not when synthesized in E . coli . We synthesized RAE1 protein both in a rabbit reticulocyte lysate system and in E . coli (not shown) and performed pull-down assays with HIS-NUP98(150–224) or GST-NUP98(150–224) purified from E . coli . Both HIS- and GST-NUP98 (150–224) bind to RAE1 (hemagglutinin [HA]-tagged) generated in a rabbit reticulocyte lysate system , but failed to bind recombinant RAE1 synthesized in E . coli (not shown). To test the folding of RAE1 purified from E . coli and in a rabbit reticulocyte lysate, we analyzed their sensitivity to tryptic cleavage . We found that tryptic cleavage of in vitro–translated [ 35 S]-methionine–labeled HA1-RAE1 removes ∼3 kD and leaves a large stable fragment of 42 kD, despite the presence of many potential cleavage sites (data not shown). By contrast, RAE1 from E . coli was extensively degraded because of cleavage at multiple tryptic sites (data not shown). This result implies that in E . coli RAE1 cannot fold into a compact, globular structure capable of interacting with NUP98. To investigate whether RAE1 and NUP98 establish direct contact, we used chemical cross-linkers . Cross-linking of residues from in vitro–translated [ 35 S]- methionine–labeled HA1-RAE1 and recombinant HIS-NUP98(150–224) or GST-NUP98(150–224) should yield specific cross-linked products of ∼56 and ∼83 kD, respectively. The predicted cross-linked products were indeed obtained with DSS , a reagent that cross-links mainly lysine residues, but not with BMH, a sulfhydryl-reactive cross-linker (data not shown). DSS-mediated coupling of RAE1 to the GLEBS-like motif of NUP98 revealed that the interaction is direct and not mediated though another protein. It has been reported that RAE1 can be UV cross-linked to poly(A) + RNA , and theoretically, binding between RAE1 and GLEBS-like motif may be established via mRNA. To investigate this possibility, we synthesized [ 35 S]-methionine–labeled HA1-RAE1 protein, removed the mRNA from the reticulocyte lysate with either Micrococcal nuclease or RNase A, and performed pull-down assays with GST-NUP98(150–224) beads. Neither the nuclease nor the RNase A treatment had any effects on the binding ability of RAE1 to NUP98 in vitro. Pull-down assays were also performed after addition of various amounts of poly(A) + mRNA isolated from HtTA cells. However, the binding efficiency of HA1-RAE1 to the GLEBS-like motif was similar irrespective of the mRNA amount present during the binding reaction . In summary, our in vitro-binding studies indicate that the interaction between RAE1 and the GLEBS-like motif of NUP98 is direct and mRNA independent. To start investigating how RAE1 binds to the GLEBS-like motif of NUP98 at the NPC, we generated a series of mutants with deletions in the NH 2 - or COOH-terminal non– WD-repeat extensions and tested them for their ability to interact with NUP98 by using a coimmunoprecipitation approach . Mutant HA1-RAE1(33–368), which lacks the entire non–WD-repeat NH 2 -terminal extension, was able to coprecipitate NUP98, although with reduced efficiency compared with wild-type RAE1 . Extension of this deletion into the first WD repeat [HA1-RAE1(66– 368)] abolished interaction with NUP98 . HA1-RAE1(1–329), which lacks the COOH-terminal 39 amino acids, again failed to coprecipitate NUP98 . By contrast, mutant HA1-RAE1(1–359), which lacks the COOH-terminal nine residues containing a highly conserved basic motif that has been shown to be essential for Rae1p function in S . pombe , could coprecipitate NUP98 . Hence, the basic motif of RAE1 may attribute a critical cellular function of RAE1 other than binding to NUP98. In nontransfected HtTA cells, RAE1 was localized prominently at the NE, but substantial amounts of RAE1 were also found in the nucleus and cytoplasm . A very similar distribution pattern was observed in HtTA cells that moderately overexpress HA1-RAE1 ; however, more robust overexpression resulted in a disproportionate increase of RAE1 levels in the nucleus (not shown). As expected, all deletion mutants but the ones that failed to coprecipitate NUP98 displayed overt NE localization when transiently expressed in HtTA cells . The above experiments suggest that both the WD repeat propeller and the COOH-terminal non– WD-repeat extension of RAE1 contribute to NUP98 binding. The HA1-RAE1(66–368) mutant suggested that the WD-repeat propeller of RAE1 is implicated in NUP98 binding. To further define the role of the WD-repeats in the RAE1–NUP98 interaction, we mutated single WD-repeats at their highly conserved aspartic acid residue positioned in the turn connecting the β strands b and c of a propeller blade . We targeted these particular residues because their conservation in 85% of all known WD-repeats indicates that they perform an important role within the propeller structure . Moreover, it has been reported that point mutations at the conserved aspartic acid residues of the WD-repeat proteins Gβ and sec13 can cause local distortions in the structure of individual propeller blades without affecting the overall structure of the propeller . We expressed the point mutants in HtTA cells and determined their ability to interact with NUP98. We found that the point mutation in propeller blade 4 , but not the point mutation in blade 2 or 3 , abolished RAE1's ability to bind to NUP98. Accordingly, HA1-RAE1-D2 and -D3 displayed a prominent NE localization when transiently expressed in HtTA cells. However, HA1-RAE1-D4 was undetectable at the NE. Together, the above studies underscore that the WD-repeat propeller is implicated in the binding of RAE1 to NUP98. Moreover, they suggest that individual WD-repeats differentially support the RAE1–NUP98 interaction. RAE1 could be permanently or transiently bound to NUP98 at the NPC, or both. As a first step to investigate whether RAE1 has dynamic properties, we microinjected in vitro–translated [ 35 S]-methionine–labeled mouse RAE1 into Xenopus oocytes and analyzed its ability to shuttle between the nucleus and the cytoplasm. The NLS-containing protein CBP80 was coinjected with RAE1 to serve as a control for nuclear protein import, and proper injection and dissection of the oocytes . We injected RAE1 into the oocyte nucleus and quantified the fraction of RAE1 appearing in the cytoplasm at various time points after injection by phosphoimager analysis. Fig. 6 , A (lanes 1–6) and B, demonstrates that ∼24% (SD ± 4%, n = 3 independent experiments) of the RAE1 molecules injected into the nucleus is present in the cytoplasm within 30 min after injection and maximal cytoplasmic levels of 31% (SD ± 4%, n = 3) are achieved within 90 min after injection. These data suggest (a) that an equilibrium between export and import has been established around 90 min after injection or (b) that the majority of the microinjected RAE1 molecules seems to be export incompetent. In addition, export of microinjected RAE1 was completely inhibited at 0°C , indicating that RAE1 export is temperature dependent and not driven by “simple” diffusion. We also investigated whether RanGTP mediates nuclear export of RAE1 by reducing the level of RanGTP via nuclear injection of Rna1p, a normally cytoplasmic GTPase-activating protein for Ran . As shown in Fig. 6 A (compare lanes 1–6 with 7–12), coinjection of 80 μM Rna1p (1–10 μM usually induces a significant inhibition in RanGTP-dependent export) did not significantly inhibit nuclear export of RAE1, while, in accordance with previous data nuclear export of NES-tagged GST substrates was dramatically reduced. As expected from its nuclear localization, RAE1 protein injected into the oocyte cytoplasm was able to migrate rapidly into the nucleus . The combination of shuttling and poly(A) + RNA-binding properties led us to hypothesize that RAE1 travels between cellular compartments as an mRNA export factor. To investigate this possibility, we stopped mRNA synthesis in HtTA cells by adding RNA polymerase II inhibitors and asked whether the subcellular distribution of RAE1 changed. After 1 h of treatment with 5 μg/ml AMD, a dose that inhibits both RNA polymerase I and II activity, the prominent RAE1 staining at the NE normally seen in HtTA cells was no longer detectable . In contrast, normal levels of RAE1 at the NE were observed in cells exposed to a dose of AMD that only inhibited RNA polymerase I activity . When we shortened the treatment with 5 μg/ml AMD from 1 h to 15 min, the drop in RAE1 levels at the NE was still detectable, suggesting that the observed effect can be an immediate early response to RNA polymerase II inhibition. HtTA cells exposed to 50 μg/ml of the RNA polymerase II inhibitor DRB (5,6-dichloro-β- d -ribofuranosylbenzimidazole) showed a RAE1-staining pattern very similar to that observed in cells exposed to a high dose of AMD . The inhibitory effect of DRB is reversible and we analyzed whether normal RAE1 levels at the NE would be restored upon reactivation of RNA polymerase II–mediated transcription. As shown in Fig. 7 E, a prominent NE staining was observed in cells cultured for 6 h in the absence of DRB after a 1-h exposure to this component. As expected, the RAE1 levels at the NE were substantially reduced after a second DRB treatment . In both AMD- and DRB-treated cells, the decrease for NE-associated RAE1 did not coincide with significant alterations in the nuclear and cytoplasmic RAE1 levels. Inhibition of RNA polymerase II activity did not affect NUP98 association with the NE , demonstrating that the absence of substantial amounts of RAE1 at the NE was not the result of NUP98 relocation. As expected, the nuclear export receptor hCRM1, which mediates export of certain viral RNAs and U snRNP, but not cellular mRNA, retained its NE localization in the presence of AMD (5 μg/ ml) or DRB (data not shown). Together, the above experiments confirm that the NE association of RAE1 is transient rather than stable. Furthermore, they support the hypothesis that RAE1 associates with the NE as part of an mRNP complex. It has been demonstrated that an AU1-tagged version of human RAE1 localizes at high levels to the nucleus and at considerably lower levels to the cytoplasm of HeLa cells . When AU1-RAE1–expressing cells were treated with RNA polymerase II inhibitors, no major changes in the distribution of AU1-RAE1 were detected. This result may seem to contradict the data presented in this report; however, it should be stressed that the effects of RNA polymerase II activity on AU1-RAE1 levels at the NE could not be evaluated because the robust nuclear staining masked the NE staining . To reevaluate the effect of RNA polymerase II inhibitors on the distribution of overexpressed RAE1, we transiently expressed HA1-RAE1 in HtTA cells. Typically, when overexpressed at low to moderate levels, HA1-RAE1 prominently localized to the NE, but significant amounts of RAE1 were also found in the nucleus and the cytoplasm . More robust overexpression of HA1-RAE1 resulted mostly in a disproportionate increase of RAE1 levels in the nucleus, which concealed the NE staining (data not shown). We determined the effect of RNA polymerase II inhibition on the distribution of HA1-RAE1 by focusing on cells with a low to moderate level of expression. As shown in Fig. 7 J, HA-RAE1 levels at the NE decreased significantly when cells were treated with 5 μg/ml AMD for 1 h, which is consistent with the results that we obtained by using nontransfected HtTA cells. When HtTA cells transiently expressing HA1-NUP98 (150–224) or HA1-NUP98(181–224) were immunostained with 12CA5 and RAE1 antibodies 24 h after transfection, we noticed a considerable reduction of RAE1 levels at the NE. As shown in Fig. 8 , A, A′, B, and B′, NE staining of RAE1 is easily detectable in nontransfected HtTA cells (nt), but not in HA1-NUP98(150–224)– or HA1-NUP98 (181–224)–expressing cells (t). By contrast, NE staining of RAE1 remained intact when HA1-NUP98(181–224)M , a mutated GLEBS-like motif that does not interact with RAE1 , or full-length HA1-NUP98 was overexpressed . In all cases, NUP98 localization at the pores appeared unchanged (data not shown). The above results suggest that overexpressed GLEBS-like motif of NUP98 acts as a dominant negative inhibitor of RAE1-NPC association by titrating RAE1 from the NPC and/or interfering with RAE1 docking to NUP98 at the NPC. To assess whether this effect is associated with changes in mRNA export, we examined the poly(A) + RNA distribution in HtTA cells expressing the NUP98 GLEBS-like motif by in situ hybridization with an FITC-labeled oligo- (dT) 50-mer probe . With this in situ hybridization protocol, alterations in nuclear poly(A) + levels can easily be detected. Hybridized cells were examined by confocal microscopy. As shown in detail in Fig. 9 F′, poly(A) + RNA was detected in both the nucleus and the cytoplasm of nontransfected HtTA cells. In situ hybridization of transiently transfected HtTA cells expressing HA1-NUP98(150–224) or HA1-NUP98(181–224) (C and C′) revealed a dramatic increase in nuclear labeling . To confirm that the strong nuclear labeling was indeed due to RNA accumulation, HtTA cells expressing HA1-NUP98(150–224) or HA1-NUP98(181–224) were incubated with RNase for 30 min before in situ hybridization. As expected, no labeling was detectable after such treatment . As an additional control that the signal detected in the nucleus is indeed mRNA, we incubated HA1-NUP98(150–224)– expressing cells for 1 h with DRB before in situ hybridization with the oligo-(dT) 50-mer probe. We quantified the nuclear signal of 20–25 cells by using confocal microscopy and the software program QUANTIFY. We compared the levels obtained with those measured in the same amount of nontreated HA1-NUP98(150–224)–positive cells. In three independent experiments, the nuclear poly(A) + signal detected in DRB-treated cells was 29, 30, and 36% lower than in nontreated cells. Thus, a proportion of the nuclear poly(A) + RNA is either exported to the cytoplasm or rapidly degraded (or both), suggesting that at least a proportion of the signal obtained with the oligo-(dT) probe represents nuclear mRNA and not just stable nuclear poly(A) + RNA . Nuclear accumulation of poly(A) + RNA induced by HA1-NUP98(150–224) or HA1-NUP98(181–224) expression typically coincided with a decrease in cytoplasmic poly(A) + RNA levels; however, considerable amounts of polyadenylated RNA were still found in the cytoplasm . This was further corroborated using an in situ hybridization procedure optimized for detection of cytoplasmic polyadenylated RNA (for details, see Materials and Methods), as is illustrated in Fig. 9 , I and I′. No nuclear build up of poly(A) + RNA was found in cells expressing the GLEBS-like motif mutant HA1-NUP98(181–224)M , which confirms that binding of endogenous RAE1 to overexpressed GLEBS-like motif is essential poly(A) + RNA accumulation. In HtTA cells overexpressing mouse RAE1 in addition to HA1-NUP98(150–224), or HA1-NUP98(181–224) nuclear accumulation of poly(A) + RNA was either not seen or hardly detectable . Thus, proper poly(A) + RNA export can take place in the presence of transiently expressed GLEBS-like motif if RAE1 levels are increased above normal. RAE1 overexpression probably restores the cellular pool of “free” RAE1 to a level required for proper nuclear mRNA export. We verified that overexpression of HA1-tagged mouse RAE1 or nontagged RAE1 (not shown) alone did not induce any measurable alterations in poly(A) + RNA distribution compared with nontransfected HtTA cells. To verify that the poly(A) + defect was not the result of a nuclear import defect, we used BHKgrβ cells. These cells express a glucocorticoid receptor–β-galactosidase fusion protein that is strictly localized to the cytoplasm. When exposed to dexamethasone, the fusion protein translocates within 30 min to the nucleus in a quantitative fashion . When we transfected BHKgrβ cells with an HA1-NUP98(150–224) construct, they accumulated poly(A) + RNA in the nucleus with no detectable effect on the import of the glucocorticoid receptor–β-galactosidase fusion protein . Although only one protein was tested, these results show that the NLS-mediated nuclear import pathway remained intact, and that the mRNA export phenotype is not likely to be the result of a general trafficking defect. Finally, we wanted to exclude that overexpression of the GLEBS-like motif induced NPC herniations similar to those seen in yeast nup116 and gle2 knockouts and nup116p (ΔGLEBS) mutant cells . To this end, we purified transiently transfected HtTA cells expressing both HA1-NUP98(150–224) and green fluorescent protein by FACS ® ( Becton Dickinson & Co.) sorting and studied their NPC integrity using an electron microscope. None of the HA1-NUP98(150–224)–expressing cells examined displayed any herniated or clustered NPCs (data not shown). Thus, the mRNA export defect is not likely to be secondary to abnormalities in NPC structure and distribution. Taken together, our results suggest a model in which a dominant negative GLEBS-like motif directly interferes with export of poly(A) + RNA from the nucleus by targeting RAE1 the NUP98-RAE1 interaction, or both. Details about the mechanism by which mRNA is exported from the nucleus remain a mystery. Here we identified and characterized an interaction between human RAE1 and NUP98, and studied its significance in mRNA export. Our studies support a model in which RAE1 is a shuttling transport factor that permits efficient export of mRNA through its ability to anchor to a GLEBS-like NUP98 motif at the NPC. Specifically, we show that: (a) RAE1 binds to the GLEBS-like motif of NUP98 through multiple domains, including the WD propeller and part of the carboxy-terminal non–WD-repeat extension; (b) the RAE1– NUP98 interaction is direct and not via another protein or RNA; (c) RAE1 has the ability to shuttle from the nucleus to the cytoplasm and that its interaction with the NE seems to be transient rather than stable; and (d) the GLEBS-like motif, when overexpressed, binds to RAE1 and inhibits poly(A) + RNA export from the nucleus, but not NLS-mediated import and NPC structure or distribution. Analysis of the dynamic properties of RAE1 by microinjection of in vitro–translated protein into Xenopus laevis oocytes revealed that RAE1 has the ability to shuttle between the nucleus and the cytoplasm in a rapid manner. Furthermore, nuclear export of RAE1 appears to be established by a temperature-sensitive, RanGTP-independent mechanism. Several studies have demonstrated that GTP-bound Ran has an essential role in nuclear RNA export. However, different RNA classes seem to depend differently on RanGTP for their export from the nucleus. Both U snRNA and tRNA export are highly sensitive towards RanGTP depletion. On the other hand, some mRNAs, such as H4 and DHFR mRNA , apparently use RanGTP-dependent as well as RanGTP-independent mechanisms for their nuclear export, whereas export of adenovirus major late transcripts and heat shock mRNAs is unaffected by the absence of nuclear RanGTP. Therefore, the RanGTP insensitivity of RAE1 export does not argue against the idea that RAE1 may be a shuttling nuclear export factor for mRNAs. Indeed, no RanGTP-binding exportin has yet been identified that is directly involved in mRNA export , and it remains possible that the effects of RanGTP depletion on mRNA export are indirect. Two additional findings reported here support RAE1's dynamic properties. First, overexpression of the GLEBS-like motif of NUP98 causes a reduction in the level of RAE1 associated with the NE. If RAE1 is permanently associated with NUP98 at the NE, overexpression of the GLEBS-like motif is expected to have no major effects on RAE1 levels at the NE. On the other hand, if RAE1's association with the NE would be transient rather than permanent, RAE1 molecules released from the NE may form a complex with the overexpressed GLEBS-like motif of NUP98. Once established, such complexes may be defective in docking to NUP98 at the NE and induce a general decline in RAE1 levels at the NE. The second finding that emphasizes the dynamic properties of RAE1 is that the level of RAE1 at the NE appears to be dependent on RNA polymerase II activity. Specifically, we observed that the amount of RAE1 at the NE dropped considerably if cells were exposed to RNA polymerase II inhibitors. When RNA polymerase II activity was restored by removal of the inhibitor, RAE1 levels at the NE returned to normal. Thus, RAE1's translocation from the nucleus to the NE may be dependent on the availability of gene transcripts generated by RNA polymerase II. It can be argued that the effect of RNA polymerase II inhibitors on the NE association of RAE1 is secondary to the depletion or relocation of one or more other cellular factors of unknown identity. Although we cannot exclude this possibility, three of our findings argue against an indirect effect: (a) the RAE1 levels at the NE drop shortly after initiation of AMD treatment (≤15 min), (b) the reversibility of the DRB-induced effect on RAE1, and (c) the observation that the inhibitors do not alter the distribution of the transport factors NUP98 and hCRM1 . While more detailed analyses need to be done, the RNA polymerase II inhibition experiments are consistent with the notion that RAE1 is a dynamic mRNA export mediator. On the surface, the observation that the association of RAE1 with the NE depends on RNA polymerase II activity seems to contradict the data from our in vitro binding studies, which indicated that mRNA is not a cofactor in the interaction between RAE1 and the GLEBS-like motif of NUP98. This apparent paradox can be explained if one assumes that docking of a transport substrate from the nucleus to the NPC follows a two-step process. In the first step, the substrate translocates from its site of assembly in the nucleus to the nuclear periphery. In the second step, the substrate anchors to the NPC. In this light, the negative effect of RNA polymerase II inhibitors on the association of RAE1 with the NE may reflect a defect not in the ability to anchor to NUP98 at the nuclear face of the NPC but rather in RAE1's translocation from the nucleus to the NPC. Guidance of RAE1 to the NPC may be a signal-mediated process relying on external export signals, perhaps provided by proteins within the mRNP transport substrate. Because so many proteins are associated with the mRNA export substrate, multiple associated proteins may provide independent signals for export of mRNP. Our most powerful evidence for a direct role of RAE1 and the RAE1–NUP98 interaction in the mRNA export pathway comes from GLEBS-like motif overexpression studies. Typically, overexpression of the GLEBS-like motif of NUP98 resulted in reduced levels of RAE1 at the NE and nuclear accumulation of poly(A) + RNA. Presuming that RAE1 is a component of mRNP , two possibilities could explain these data. It seems fair to argue that RAE1 associates with mRNP since RAE1 and poly(A) + RNA can be cross-linked by UV irradiation in vivo . Thus, at least part of the cellular RAE1 pool is in close proximity to mRNA, although it remains to be determined whether RAE1 and mRNA interact directly (RAE1 sequences lack similarity to any known mRNA-binding motifs), or indirectly via one of the mRNA-associated proteins. In the first possibility, one might propose that the NUP98 GLEBS-like motif binds to an RAE1 molecule that is in a complex with mRNP. The presence of the GLEBS-like motif in the resulting complex would then prevent its anchoring to full-length NUP98 at the NPC. In the second possibility, one might argue that the GLEBS-like motif associates with free RAE1 protein to affect its binding to mRNP. Lack of RAE1 within the mRNP particle would then affect its proper anchoring to the NPC. We found that cells that co-overexpress the GLEBS-like motif together with full-length RAE1 displayed neither reduced levels of RAE1 at the NE nor accumulation of poly(A) + RNA in the nucleus, indicating that the mRNP export defect induced by the GLEBS-like motif is not an overexpression artifact, but rather a result of impaired RAE1 function. How could RAE1 overexpression neutralize the mRNA export defect induced by the GLEBS-like motif? If the GLEBS-like motif indeed interacts only with RAE1 that is bound to mRNP, as proposed in possibility one (above), then RAE1 overexpression is expected to have little or no effect on the formation of anchoring-defective complexes. However, if the GLEBS-like motif only associates with a free RAE1 molecule, as outlined in possibility two, then RAE1 overexpression could simply titrate GLEBS-like motifs and restore the pool of free RAE1 to a level sufficient for proper mRNP export. Thus, the data from our co-overexpression studies are apparently more consistent with possibility b than a. Additional data presented in this report are consistent with the idea that RAE1 and the RAE1–NUP98 interaction directly serve in the nuclear export pathway for mRNA. First, NUP98 fragments with point mutations in the GLEBS-like motif that abolish its association with RAE1 failed to inhibit poly(A) + RNA export from the nucleus. Thus, there is a correlation between nuclear accumulation of poly(A) + RNA and binding of the GLEBS-like motif to RAE1. Second, there is no evidence to suggest that the observed poly(A) + RNA accumulation phenotype results from impaired NLS-mediated protein import, loss, clustering, or herniation of nuclear pores, or release of NUP98 from the NE. In particular, the absence of any gross defects in NPC structure and distribution is of importance with respect to earlier work in the yeast system. In gle2 mutant yeast, as well as gle2 or nup116 null alleles , a block in poly(A) + RNA export is always accompanied by severe clustering and herniation of the nuclear pores. Because of these NPC perturbations, it has been difficult to ascertain whether Gle2p and Nup116p are mediators of RNA transport. Nup116 (ΔGLEBS) mutants , which are defective in the docking of Gle2p to Nup116p at the NPC, display spatio-structural NPC defects very similar to gle2 or nup116p mutants. Because of these defects, it is difficult to study the export function of the Gle2p– Nup116p interaction in the nup116 (ΔGLEBS) mutants. An interesting difference between S . cerevisiae Gle2p and S . pombe Rae1p is that a lack of Rae1p function results in poly(A) + RNA accumulation in the absence of any detectable NPC defects . It is conceivable that Gle2p and Rae1p functions have diverged, and that Gle2p may perform separate functions in both mRNA transport and NPC structure. Insight into how Gle2p and Rae1p function in the pathway for nuclear export of mRNA is required to address such issues. In S . cerevisiae , it will be important to establish whether the Nup116p–Gle2p interaction at the NPC serves directly in the RNA export pathway. Perhaps GLEBS-overexpression studies similar to those presented in this paper can yield such information. To gain insight into how Rae1p functions in fission yeast, a crucial step will be to identify whether S . pombe contains an S . cerevisiae Nup116p homologue that has a GLEBS-like binding site, and, if so, whether this site directly functions in mRNA export. Could RAE1 serve as the exportin for mRNA or as a factor that provides the NES signal to mRNA ? Unlike Rae1p in S . pombe , Gle2p in S . cerevisiae is not a strictly essential protein indicating that mRNA export is impaired rather than completely blocked in the absence of functional Gle2p. Thus, Gle2p does not seem to operate as the sole exportin or NES-containing factor for mRNA export. Instead, Gle2p and its homologues in fission yeast (Rae1p) and mammals (RAE1) more likely participate in mRNA export as mRNP-interacting proteins necessary for efficient anchoring of the transport complex to the NPC. RAE1 is a member of the superfamily of WD-repeat proteins. Given the high conservation of the WD-repeats, it is likely that they all fold into a propeller structure . Members of this family do not have an immediately obvious common function. Rather, the common thread between WD-repeat proteins seems to be that they make up part of large macromolecule assemblies. The capacity to assemble multiple proteins may be an essential part of their function, including that of RAE1 . The signal-transducing WD-repeat protein Gβ binds to Gγ to form a heterodimer that in turn interacts with Gα or a variety of different effector proteins . The various interactions with Gβ are established through unique as well as overlapping contact sites involving specific blades of the Gβ propeller . Similarly, the blades of the RAE1 propeller might serve as contact sites for multiple distinct RAE1 partners. This idea has some support from the observation that the interaction of RAE1 and the GLEBS-like motif of NUP98 is highly sensitive to a point mutation in blade 4, but not those in blades 2 and 3. Additionally, studies in S . pombe have demonstrated that a conserved 10-residue basic motif at the carboxy terminus of Rae1p is necessary for Rae1p function. Here, we found that this motif is not essential for the RAE1–NUP98 interaction, which is in keeping with the idea that RAE1 may be involved in an additional protein–protein interaction. A future goal to provide further insight into the complex mechanism of mRNA export from the nucleus is to determine whether RAE1 preferentially interacts with specific kinds of mRNA molecules and to define how RAE1 interacts with mRNP particles. Preliminary in vivo cross-linking studies suggest that RAE1 indeed interacts with at least one other protein besides NUP98. | Study | biomedical | en | 0.999999 |
10209022 | The following proteins were expressed in Escherichia coli BLR/Rep4 and purified as previously described: Xenopus importin α , human importin α , NTF2 , His-tagged RanQ69L and Ran wild type , Rna1p , RanBP1 , and CAS . Snurportin 1 was cloned into the BamHI-XmaI sites of pQE30 (Qiagen), expressed with an NH 2 -terminal His tag and purified on nickel-NTA agarose followed by dialysis in 20 mM Hepes-KOH, pH 7.5, 200 mM NaCl, 2 mM magnesium acetate, 250 mM sucrose. GST-snurportin 1 was expressed from pGEX T4 and purified on glutathione Sepharose 4B, followed by chromatography on MonoQ. zz-snurportin 1 (full length and fragments) were cloned into the NcoI-BamHI sites of pQE60 and expressed with a COOH-terminal His tag and with NH 2 -terminal IgG binding domains from Staphylococcus aureus protein A. Human CRM1 was cloned into the NdeI-BamHI sites of pET3a and was expressed in BL21 DE3 without addition of IPTG at 30°C. This recombinant CRM1 is untagged and was purified on nickel-NTA agarose followed by chromatography on MonoQ. The cDNA coding for the HIV Rev protein was cloned into the NcoI-BamHI site of the 6z60 vector . Expression was with an NH 2 -terminal 6z tag and a COOH-terminal his tag, purification was on nickel agarose. Labeling of snurportin 1 with fluorescein 5′ maleimide ( Calbiochem ) was performed at a 1:1 molar ratio in 50 mM Hepes-KOH, pH 7.5, 200 mM NaCl, 2 mM magnesium acetate, 250 mM sucrose for 2 h on ice. Free label was removed on a NAP5 column equilibrated with 50 mM Hepes-KOH, 150 mM NaCl, 2 mM magnesium acetate. Labeling of Xenopus importin α has been described before . Antibodies against the following antigens have been described before: human importin β , Xenopus RanBP7 , and human CAS . Anti-CRM1 antibodies were raised in rabbits against a CRM1 peptide (cys-EKHKRQMSV). Antibodies were affinity-purified on sulfoLink (Pierce) to which the antigens had been coupled. Permeabilization of HeLa cells and nuclear import reactions in suspension was performed essentially as described before . The import buffer contained: 2 mg/ml nucleoplasmin core (to block nonspecific binding), 20 mM Hepes-KOH, pH 7.5, 140 mM potassium acetate, 5 mM magnesium acetate, 250 mM sucrose, 0.5 mM EGTA. Reactions were supplemented with an energy-regenerating system (0.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, 50 μg/ml creatine kinase), 5 μM RanGDP, and 1 μM NTF2. zz-tagged RanQ69L and snurportin 1 prebound to IgG-Sepharose were used as affinity matrices. The z domain is the IgG binding domain from S . aureus protein A. 500 μl of cytoplasmic HeLa extract or 200 μl of E . coli lysate expressing the recombinant proteins was incubated with 20 μl of affinity matrix for 4 h at 4°C in binding buffer (50 mM Hepes-KOH, pH 7.5, 200 mM NaCl, 5 mM magnesium acetate, 0.005% digitonin). The beads were recovered by mild centrifugation and washed three times with 1 ml binding buffer. Elution was with 100 μl of 50 mM Tris-HCl, pH 7.5, 1 M magnesium chloride for 10 min at room temperature. Proteins were precipitated with 90% isopropanol (final concentration), dissolved in SDS sample buffer, and analyzed by SDS-PAGE. Kinetic measurement of the RanGTPase was as described before with modifications described in the figure legends. The m 3 G-cap (m3GpppAmpUmpA-oligonucleotide) was described previously and the m 7 GpppG-cap dinucleotide was purchased from Pharmacia . Oocyte injection and analysis of microinjected RNA by denaturing gel electrophoresis and autoradiography were performed as described . The mutant RNAs used (U1ΔSm, U5ΔSm) lack protein-binding sites required for the nuclear import of these RNAs and thus remain in the cytoplasm after export from the nucleus. Snurportin 1 mediates the m 3 G-cap–dependent import of U snRNAs , most likely in conjunction with importin β . To achieve multiple rounds of import, snurportin 1 has to return to the cytoplasm after delivering its import substrate to the nucleus. Since importin α and snurportin 1 both contain an IBB domain for binding to and import by importin β, it was of interest to know if snurportin 1 would also exit the nucleus in the same way as importin α, by using CAS for reexport . Surprisingly however, we could not detect any interaction between CAS and snurportin 1 (see below), suggesting that snurportin 1 export is mediated by a distinct factor. To identify this factor, we immobilized snurportin 1 and tested which proteins from a HeLa extract it would bind . Without further addition, three major bands were recovered in the bound fraction and identified by Western blotting as importin β, CRM1, and importin 7 (formerly called RanBP7). The binding of importin β to snurportin 1 was expected from previous studies. Importin 7 was apparently recovered only because it binds importin β . Binding of the export receptor CRM1 to snurportin 1 might suggest that CRM1 mediates export of snurportin 1. Indeed, the pattern of snurportin 1–bound proteins changed when RanQ69L GTP was included in the binding reaction. This Ran mutant is GTPase-deficient, remains GTP-bound even in the presence of RanGAP1, and can therefore be used to mimic a nuclear environment in a cytoplasmic extract. Under these conditions, the binding of the two importins was greatly reduced. In contrast, binding of CRM1 was enhanced by the presence of RanGTP. Thus, CRM1 behaved as expected for a snurportin-specific export receptor. The interaction between snurportin 1 and CRM1 could be either direct or mediated by an unidentified factor in the HeLa extract. To exclude the second possibility we repeated the binding experiments using E . coli lysates containing recombinant transport receptors. CRM1 bound to immobilized snurportin 1 , and this binding was enhanced by the addition of RanQ69L. Ran was also recovered in the bound fraction, indicating the formation of a trimeric snurportin/CRM1/RanGTP complex . Conversely, the trimeric complex could also be assembled using immobilized RanQ69L, free CRM1, and GST-snurportin 1 . The high cooperativity of complex formation was evident from the observation that CRM1 binding to RanGTP was not detectable in the absence of snurportin 1 but was strong in its presence. To obtain more quantitative data on the formation of the trimeric complex, we used a kinetic assay . Binding of an exportin to RanGTP prevents GTPase activation by RanGAP1. GTP hydrolysis can easily be quantified and used to calculate the proportion of Ran that is exportin-bound. From the dose dependence of the effects one can estimate apparent dissociation constants ( K d ) for the complex formation. Without further addition, CRM1 bound RanGTP only very weakly ; In the presence of a saturating concentration of snurportin 1 (2 μM), however, the K d shifted to 4–5 nM, emphasizing the highly cooperative nature of the snurportin/CRM1/RanGTP complex formation. The specificity of the effect was verified by two controls. First, the presence of importin α had no effect on the RanGTP/CRM1 interaction. Second, CAS responded only to its specific export substrate importin α, but not to snurportin 1 . Export complexes form in the nucleus and need to be disassembled after export in the cytoplasm. In the case of CAS and exportin-t, this disassembly is brought about by the concerted action of RanBP1 and RanGAP1. However, there is a complication in respect to CRM1, namely that RanBP1 and RanGAP1 apparently also contain leucine-rich NESs and are thus also potential export substrates for CRM1 which could stabilize a CRM1/RanGTP complex. Therefore, it was quite surprising that RanBP1 rendered also the snurportin/CRM1/RanGTP complex sensitive to RanGAP and thereby caused disassembly . The observation that CRM1 binds snurportin 1 with high affinity and specificity strongly suggests that CRM1 mediates nuclear export of snurportin. To test this directly, we performed the pulse-chase export experiment shown in Fig. 4 . Nuclei of permeabilized HeLa cells were first loaded for 10 min with fluorescein-labeled snurportin 1 and Texas red–labeled importin α in the presence of importin β, Ran, NTF2, and an energy-regenerating system. The sample was then split into four. The first aliquot was fixed immediately and confocal fluorescence microscopy confirmed that snurportin 1 and importin α had both efficiently accumulated in the nuclei. The three remaining aliquots were supplemented with either CRM1, CAS, or buffer and incubated for another 10 min before these samples were fixed also. The addition of CRM1 caused a significant depletion of the nuclear pool of snurportin and an increased NPC signal that probably represented import and export intermediates. However, CRM1 did not change the localization of importin α. Addition of CAS had exactly the opposite effect. It led to nuclear export of importin α but had no effect on snurportin. Therefore, we can conclude that CRM1 specifically mediates export of snurportin. The experiment also confirms our previous data that CAS is the importin α–specific exportin and contradicts the suggestion by Boche and Fanning that importin α would exit the nucleus by virtue of a Rev-like NES, i.e., in a CRM1-dependent manner. Productive import cycles require snurportin 1 to enter the nucleus with cargo and to exit without cargo. Although nuclear retention of the import substrate might contribute to this directionality, transport would be most efficient if the affinity of snurportin 1 for m 3 G-capped U snRNPs was high during nuclear entry and low during reexport. β-Family transport receptors use RanGTP to regulate interactions with their transport substrates. This mechanism is not available to snurportin 1 because it does not bind Ran . However, it uses importin β for nuclear entry and CRM1 for exit from the nucleus and the binding of the two transport receptors might regulate snurportin's affinity for the m 3 G-cap. Therefore, we tested for a cross-talk between the binding sites for the m 3 G-cap and CRM1. In the absence of RanGTP, CRM1 bound weakly to snurportin 1 . A 10-fold excess of a m 3 G-cap oligo prevented CRM1 binding completely, suggesting that m 3 G-cap and CRM1 binding to snurportin 1 are mutually exclusive. However, the m 3 G-cap oligo could only reduce, but not prevent, CRM1 binding in the presence of Ran-GTP, i.e., when the exportin was in its high affinity form. The antagonism of CRM1 and m 3 G-cap binding was also clearly evident in the kinetic assay that measures snurportin/CRM1/RanGTP complex formation . The presence of 5 μM m 3 G-cap shifted the equilibrium and reduced the apparent affinity of snurportin 1 for the CRM1/RanGTP complex ∼40-fold. The effect was highly specific as verified by two controls. First, a m 7 G-cap analogue, which does not bind snurportin 1, had no effect on the snurportin/CRM1/RanGTP interaction. Second, the m 3 G-cap did not interfere with the interaction between CRM1 and BSA conjugated to HIV Rev NES peptides and is thus snurportin 1–specific . A mutual displacement of CRM1 and m 3 G-cap from snurportin 1 should occur twice per import cycle. When the trimeric export complex has been exported and RanGTP removed, a still quite stable CRM1/snurportin 1 dimer ends up in the cytoplasm. Binding of the import substrate to snurportin 1 displaces the low affinity form of the exportin . Conversely, following nuclear entry, the U snRNPs need to be displaced from snurportin 1 by the high affinity form of CRM1 in the presence of RanGTP. Because the m 3 G-cap binds snurportin 1 very tightly, displacement would only be efficient if the CRM1/RanGTP complex would bind with at least comparable affinity. In fact, Fig. 5 D shows that CRM1 binds snurportin 1 50 times more tightly ( K d ≈ 10 nM) than the HIV Rev protein ( K d ≈ 0.5 μM). The difference is even more dramatic when compared with the isolated NES from HIV Rev. A peptide comprising the Rev activation domain plus a few flanking residues bound CRM1 700 times weaker ( K d ≈ 7 μM), and the minimum Rev activation domain itself even 5,000-fold weaker ( K d ≈ 50 μM) than snurportin 1. The 5,000-fold higher affinity of CRM1 for snurportin 1 compared with the affinity for the minimum Rev activation domain (Rev-NES) raised the question as to the nature of snurportin's export signal. Snurportin 1 contains an IBB domain for importin β binding (residues 26–65), while m 3 G-cap binding has been attributed to the part of the protein that is COOH-terminal to the IBB . Analysis of the amino acid sequence of snurportin 1 did not reveal the existence of a single amino acid sequence matching the consensus leucine-rich NES; rather, several regions were found which distantly resemble it. To delineate the binding site for CRM1 on snurportin 1 we expressed NH 2 - and COOH-terminal snurportin 1 deletion mutants in E . coli , immobilized them, and tested their capacity to bind CRM1 in the presence or absence of RanQ69L . As a control, we tested the same mutants for binding to importin β and found, as expected, that all deletion mutants containing the IBB domain (residues 26–65) also bound importin β . Deletion of as few as 26 amino acids from the NH 2 terminus prevented CRM1 binding and also nuclear export in the permeabilized HeLa cell assay . A snurportin 1 fragment lacking 74 amino acids from the COOH terminus (residues 1–285) retained the ability to bind CRM1 in the solution binding assay ; however, the kinetic assay indicated that the deletion lowered the affinity for CRM1 by 60% (not shown). A smaller fragment comprising residues 1–159 lost CRM1 binding entirely. Therefore, it appears that binding to CRM1 is sensitive to deletions from both the NH 2 and the COOH termini. Thus, in contrast to the short export signals characterized previously, snurportin 1 uses a large domain of at least 159 residues, but probably >285 residues, to bind CRM1. Such a domain could make more contacts to CRM1 and thereby bind more tightly than a simple NES. However, the main advantage might be that a conformational change in this domain can allow regulation of affinity. We next wanted to know if snurportin 1 would also use CRM1 for reexport to the cytoplasm in Xenopus oocytes. As an initial experiment, we found that snurportin 1 indeed gets rapidly exported when injected into nuclei of Xenopus oocytes (not shown). If this rapid export is mediated by CRM1, then one would expect that snurportin 1 also competes other CRM1-mediated export pathways. To test this, we injected a mixture of 32 P-labeled RNAs consisting of DHFR mRNA, histone H4 mRNA, U1ΔSm RNA, U5ΔSm RNA, U6Δss RNA, and tRNA i Met into nuclei of Xenopus oocytes. After 180 min, the mRNAs, the U1 and U5 RNAs, and the tRNA had been efficiently exported to the cytoplasm . U snRNA export is known to require CRM1 and when GST-snurportin 1 was coinjected, export of U1 and U5 was severely inhibited, but export of tRNA and the two mRNAs remained unaffected. Thus, one can conclude that snurportin 1 accesses the CRM1 export pathway also in this cell type. Furthermore, the very high affinity for CRM1 should make snurportin 1 a quite useful tool to characterize export of other substrates, and sensitivity towards competition by snurportin 1 should be a stringent test for a CRM1 requirement. Transport between nucleus and cytoplasm appears to be largely mediated by adapter molecules and Ran-binding transport receptors that shuttle between the two compartments. Multiple rounds of import require a cyclic process that results not only in the transport of the cargo to the other side of the nuclear membrane, but also in the subsequent restoration of the original state of the transport system (recycling). We have investigated here a recycling reaction for U snRNP import, namely the reexport of snurportin 1 back to the cytoplasm . Snurportin 1 binds m 3 G-capped U snRNP in the cytoplasm and is imported by importin β into the nucleus . There, importin β binds RanGTP, which destabilizes the interaction with snurportin 1 . Interestingly, the snurportin/importin β/RanGTP complex is considerably more stable than the corresponding complex with importin α and can even be isolated in small amounts . This could indicate that the actual dissociation of snurportin 1 from importin β does not occur immediately after NPC passage, but further inside the nucleus. Snurportin 1, then, has to release the m 3 G-capped import substrate before it can form a trimeric export complex with CRM1 and RanGTP. The formation of this complex is highly cooperative and we estimate that snurportin 1 increases the affinity of CRM1 for RanGTP ∼1,000 fold . The export complex would then be transferred to the cytoplasm, where it must be disassembled. This disassembly apparently occurs in several steps, involves GTP hydrolysis, and requires RanBP1 and RanGAP1 . CRM1-bound RanGTP resists GTPase activation by RanGAP. Therefore, RanBP1 probably causes an initial release of Ran in the form of a RanBP1/ RanGTP complex in which the RanGTPase can be activated by RanGAP1 . GTP hydrolysis is the irreversible step of the disassembly because RanGDP cannot rebind to CRM1. Removal of RanGTP could alternatively be accomplished by the RanBP2/ SUMO-RanGAP1 complex that is located at the cytoplasmic filaments of the NPC . Although Ran-free CRM1 is the low affinity form, the remaining CRM1/snurportin 1 heterodimer is still quite stable. However, binding of an m 3 G-capped substrate displaces CRM1 . The snurportin/importin β complex would then form and mediate import of the next U snRNP. For productive transport cycles, carrier molecules need to have different affinities for their substrates during nuclear entry and exit. Ran-binding transport receptors can regulate their affinity for cargo with the aid of the RanGTPase. Adapter molecules do not directly bind RanGTP and, instead, they apparently use differential binding of transport receptors to coordinate the interaction with their cargoes. The first example for that has been import by importin α. Binding of importin β on the way into the nucleus increases the affinity importin α for the NLS . Conversely, CAS preferentially binds and exports NLS-free importin α . A second precedence is the nuclear CBC which binds 7 monomethyl-cap structures and promotes export of m 7 G-capped RNAs . After export, CBC needs to release the RNA before it is reimported by the importin α/β heterodimer. CBC can simultaneously bind a m 7 G-cap and importin α. However, binding of importin β to the importin α/CBC/ RNA complex causes the displacement of the RNA, allowing CBC to reenter the nucleus without its export substrate . Here we show that the affinity of snurportin 1 for its import substrate is regulated by CRM1. The molecular basis for the mutual exclusivity of CRM1 and m 3 G-cap binding to snurportin 1 is still unknown. The two binding sites in snurportin 1 are not identical because the 65 NH 2 -terminal residues are dispensable for m 3 G-cap binding but are required for the interaction with CRM1 . However, the binding sites could overlap and thereby exclude simultaneous binding. Alternatively, snurportin 1 might adopt two distinct conformations, one with a high affinity for a m 3 G-cap, but weak CRM1 binding, and the other with the opposite preference. U snRNP release from snurportin 1 could occur spontaneously and rebinding could be prevented by CRM1. Alternatively, the release would be more efficient if CRM1 could actively displace m 3 G from snurportin, i.e., not only shift the equilibrium, but also increase the off-rate. In addition, it should be noted that formation of spliceosomes would also retain the mature U snRNP in the nucleus. Since its discovery as the receptor for leucine-rich NESs , CRM1 has been implicated in nuclear export of a number of substrates including MAPKs , cyclin B1 , NFAT , actin , Dsk1p , and Pap1p . The involvement of CRM1 in the export of the majority of these substrates was suggested by the identification of a leucine-rich NES and/or their sensitivity to the drug leptomycin B, a CRM1 inhibitor . A direct physical interaction between CRM1 and its potential export substrates has not always been demonstrated and it is still possible that additional bridging factors are involved. Apart from mediating the export of RRE-containing viral RNAs through Rev, CRM1 is also believed to participate in the export of U snRNPs and possibly in mRNA export in yeast . Therefore, it appears that from the three exportins identified so far CRM1 has the broadest range of substrates. In contrast, CAS and exportin-t appear to have each specialized in the export of a single extremely abundant class of export substrates, the members of the importin α family and tRNA, respectively. The question of whether CRM1 is involved in mRNA export in higher eukaryotes has been a controversial issue recently . In contrast to the experimental evidence against a role of Crm1p in mRNA export provided by Fischer et al. and Fornerod et al. , a later study arrived at the opposite conclusion . The fact that mRNA export from Xenopus oocytes nuclei resists competition by snurportin 1 confirms the conclusions by Fornerod et al. and Fischer et al. and makes it very unlikely that mRNA export in higher eukaryotes is mediated by CRM1. CRM1 substrates other than snurportin 1 have been suggested to interact with CRM1 by virtue of a leucine-rich stretch of 8–10 amino acids, a prototype being the Rev activation domain . Surprisingly, however, the isolated activation domain binds CRM1 100 times weaker than the full-length Rev protein . This could have two explanations. First, a leucine-rich export signal might need an appropriate protein context to adopt the conformation required for high-affinity CRM1 binding. In addition, our data indicate that residues flanking a given NES can also significantly contribute to its affinity for CRM1 . CRM1 binds snurportin 1 still 50-fold stronger than the full-length Rev protein. The probable reason for this difference is that CRM1 has to displace the avidly binding m 3 G-capped import substrate. The structural basis for the high affinity of the snurportin/CRM1 interaction is still unclear, but nearly the entire snurportin 1 molecule appears to be required for it. The amino acid sequence of snurportin 1 does not contain a single perfect match to the consensus leucine-rich NES, but several regions that appear to resemble it. The affinity might be that high because several of these imperfect NESs interact synergistically with the same CRM1 molecule. However, the interaction of CRM1 with snurportin 1 might also be completely different from that with the prototype NES. This would not be the first case for a transport receptor recognizing very different signals. Transportin, for example, can bind the M9 import signal, which does not have a single essential basic residue, and it also binds the extremely basic import signal from the ribosomal protein L23a . | Study | biomedical | en | 0.999997 |
10209023 | Rabbit reticulocyte lysates were prepared and treated with micrococcal nuclease ( Calbiochem-Novabiochem Corp. ) as described in Jackson and Hunt . Dr. D. Melton (Harvard University, Cambridge, MA) provided the SP6 RNA polymerase expression plasmid pSP64T . Edeine was the gift of Dr. H. Lodish (Massachusetts Institute of Technology, Cambridge, MA). SP6 RNA polymerase and RNase inhibitor (RNASIN) were from Promega Corp. T7 RNA polymerase was from New England Biolabs . [ 35 S]Methionine (Translabel, 1,000 Ci/mMol) was obtained from New England Nuclear . Apyrase and soybean trypsin inhibitor were from Sigma Chemical Co. Rabbit polyclonal anti-PFD subunit 6 (PFD 6) was described previously . A purified rat mAb, 23C, recognizing TCP-1α, was used for the analysis of CCT . Purified rabbit skeletal muscle actin was obtained from Cytoskeleton Inc. AffiGel-10 was from Bio-Rad. DNase I was from Worthington Biochemicals. The sequence at the initiator methionine codon of chicken βII-tubulin was modified to create a NcoI site by Dr. H. Sternlicht (Case Western Reserve University, Cleveland, OH). The β-tubulin, human β-actin , and Aequorea victoria green fluorescent protein coding sequences were shuttled into the pSP64T derivative, pSPBP4, replacing the preprolactin gene. pSPBP4 contains the SP6 promoter, the β-globin 5′ untranslated region, an NcoI site encoding an initiation codon with an optimal Kozak consensus sequence , the gene for bovine preprolactin, and a polylinker, in that order. The expression plasmids used to produce yeast cytosolic invertase, α-tubulin, and firefly luciferase (pGEM-luc) mRNAs were described previously . DNA templates were transcribed after complete linearization with the appropriate restriction enzymes to create full-length and the desired truncated coding regions. G ppp G capped mRNA templates generated by in vitro transcription using SP6 RNA polymerase as described previously were used without purification. Transcription with T7 RNA polymerase was performed in 40 mM Tris-HCl, pH 7.9, 2 mM spermidine, 10 mM DTT, 12.4 mM MgCl 2 , 2 mM each of ATP, CTP, UTP, and G ppp G, and 0.4 mM GTP at 37°C for 90 min. To promote full-length transcription, the GTP concentration was then increased to 2 mM and the incubation continued for 15 min. T7 transcripts were precipitated with ethanol and resuspended in diethylpyrocarbonate (DEPC) treated water. Rabbit reticulocyte translation reactions (40% lysate) were performed at 22–24°C for the times indicated in 20 mM Hepes, pH 7.4, 100 mM KCl, 2 mM MgCl 2 , 2 mM dithiothreitol, 0.2 mM spermidine, and 10 mM S-adenosyl-methionine. In the experiment shown in Fig. 3 , edeine (10 mM) and 7-methylguanosine monophosphate (7- Me GMP; 4 mM) were added as follows: after 5 min of translation for −mRNA (no added mRNA) and 99 aa actin; after 6 min for 123 aa, 145 aa, 156 aa, and 257 aa actin; and after 9 min for 336 aa and full-length actin. Aliquots of the reactions were removed for analysis as follows (e, early; l, later): e = 8 min, l = 20 min for −mRNA; e = 8 min, l = 12 min for 99 aa actin; e = 9 min, l = 16 min for 123 aa actin; e = 9 min, l = 20 min for 145 aa and 156 aa actins; e = 12 min, l = 25 min for 257 aa actin; e = 12 min, l = 70 min for 336 aa and full-length actins. The reticulocyte lysate was programmed with mRNA encoding the 336– (or 257–) amino acid NH 2 -terminal fragment of actin and incubated at 24°C for 8 min. Edeine (10 mM) and 7- Me GMP (4 mM) were added to inhibit new chain initiation. The reaction was further incubated for 9 min (7 min for the 257 amino acid actin) to allow for complete translation of the mRNA template. The nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, ATP levels were depleted by incubation for 10 min at 24°C with apyrase (50 U/ml) and 25 mg/ml soybean trypsin inhibitor. The mixture was applied to a gradient of sucrose consisting of 0.2 ml of 68% sucrose overlayered with 0.5 ml 20% sucrose, both containing 20 mM Hepes, pH 7.4, 2.1 mM MgCl 2 , 100 mM KCl, 2 mM reduced glutathione, 0.1 mM EDTA, and 0.2 mM cycloheximide. The gradient was centrifuged in a Beckman TLA100 SW55 ultracentrifuge rotor at 50,000 rpm for 90 min at 4°C. After centrifugation, the gradient was fractionated from the top. An aliquot of each gradient fraction was incubated for 10 min with 0.5 mM puromycin and 20 μg/ml RNase A, and then analyzed by Tricine SDS-PAGE. RNA was isolated from each of the gradient fractions by extraction with phenol/chloroform and precipitation with ethanol. The RNA samples were suspended in DEPC-treated water and denatured by incubation at 60°C in 7 M urea RNA sample buffer. The RNA was fractionated by 5% PAGE containing 7 M urea, the gel stained with ethidium bromide, and the UV transilluminated image photographed. The tRNA and high molecular weight ribosomal RNAs recovered from each fraction were quantified using NIH Image software. For native-PAGE analysis, aliquots of the starting materials applied to the gradient and of each gradient fraction were incubated with puromycin (0.5 mM), apyrase (50 U/ml), and soybean trypsin inhibitor (25 mg/ml) for 10 min at room temperature, followed by digestion with RNase A (20 μg/ml) in native-PAGE sample buffer for 10 min before analysis by native-PAGE as described below. SDS-PAGE analysis was performed using the gel formulations of Blattler et al. or when indicated, a Tris/Tricine system . When peptidyl-tRNA species were to be examined, the translation products were heated in Laemmli sample buffer, pH 6.8 . tRNA free polypeptides were analyzed after the peptidyl-tRNA species were dissociated by incubation with 0.5 mM puromycin at room temperature for 10 min, then heated in SDS-PAGE loading buffer, pH 11.3. The discontinuous gradient native gels used were composed of two or three layers . The upper chamber (cathode) buffer consisted of 50 mM Tricine and 15 mM BisTris, pH 7.0. The lower chamber (anode) buffer chamber contained 50 mM BisTris, pH 7.0. Reaction mixtures were adjusted with native gel loading buffer to final concentrations of 50 mM BisTris, pH 7.0, 5% glycerol, 2 mM reduced glutathione, 1 mM each methionine and cysteine, and a small amount of tracking dye, Ponceau S. The gels were run initially at 50 V for 20 min (∼5 mA), and then with a maximum current of 13 mA at 4°C until the tracking dye migrated to the bottom of the gel. The position of migration of native molecular mass standards is indicated in each figure: soybean trypsin inhibitor, 21 kD; BSA monomer, 70 kD; BSA dimer, 130 kD; BSA trimer, 200 kD; ferritin, 450 kD; and α2-macroglobulin, 780 kD. Proteins were visualized either by Coomassie blue staining or by fluorography. Radiolabeled proteins (where indicated) were quantified by scanning the x-ray film image and processing with NIH Image software. Positive identification of components present within nascent chain–chaperone complexes was performed by an electrophoretic mobility shift assay with the nascent chain serving as the radiolabeled probe. Antibodies to the chaperone protein of interest or control antibodies (e.g., preimmune serum) were added to the puromycin-released and ATP-depleted reticulocyte lysate translation products. After incubation for 15–20 min at room temperature, native gel sample buffer was added and the samples analyzed by native-PAGE. The puromycin-released and ATP-depleted reticulocyte lysate translation products were mixed with antibodies (preimmune, anti–PFD 6, or anti–TCP-1α) previously bound to protein A–Sepharose or immobilized DNase I in native gel sample buffer containing 100 μg/ml BSA. After 45 min at 4°C with occasional mixing, the Sepharose beads were removed by centrifugation and the supernatant solution was analyzed by native-PAGE. Sepharose beads were washed four times in PBS, the bound proteins eluted by heating in Laemmli sample buffer, and analyzed by SDS-PAGE. In vitro translated actin was incubated in the reticulocyte lysate supplemented with 400 μg/ml of DNase I at room temperature for 15 min. Native-PAGE loading buffer was added and the mixture analyzed by native-PAGE. Translation reaction products were fractionated by native-PAGE as described above. The lanes were excised from the gel and incubated for 10 min in two times concentrated Laemmli sample buffer at 60°C. The entire gel lane then was applied to and electrophoresed through a second dimension of 12.5% SDS-PAGE. The pathway of actin maturation was investigated using translation in vitro. Full-length actin mRNA was translated in the presence of [ 35 S]methionine and the reaction products analyzed by both SDS-PAGE and native-PAGE. In the first experiment, full-length actin mRNA was translated for 12 min. Inhibitors of translational initiation (edeine and 7- Me GMP) were added and the translation reactions continued to allow for polypeptide chain elongation and completion. At various times, aliquots were removed and incubated with apyrase (to deplete ATP levels and thereby stabilize chaperone–nascent chain complexes) and puromycin (to release unfinished nascent chains from the ribosome). Analysis of the reaction products by SDS-PAGE revealed a small amount of full-length actin after 12 min of translation . Maximal amounts of full-length actin had accumulated between 20 and 30 min. Native-PAGE analysis showed that these reaction products consisted of full-length monomeric actin together with at least four actin-containing complexes with apparent masses between 150 and 800 kD . The monomeric actin species had acquired its native folded state as evidenced by its ability to interact with DNase I, an assay used to monitor the folding status of actin . Quantitation of the different actin complexes accumulating over time was performed and is presented in two different ways in Fig. 1 , B and C. Such analyses showed that the ∼150-kD complex (species I) was the first complex formed. Subsequently, the ∼800-kD complex (species II) was observed to increase. Finally, species III and IV were found to form later in time. Based on its apparent mass of ∼800 kD, species II represents actin bound to the CCT, which has been described by others. The nature of the other complexes, in particular species I, is not known and is the major focus of the present study. Note that by 40 min the levels of both species I and II had markedly diminished, and that full-length monomeric actin was now the predominant species. Curiously, the levels of the ∼500-kD complex did not change significantly over the course of the last 20–30 min of translation. Given their low levels and relatively late appearance (i.e., they rise only after the native full-length actin monomer is present), it is unlikely that species III and IV represent intermediates in the pathway of actin monomer folding. Hence, we will restrict our focus here to the characterization of species I and II. In a second experiment of this type, full-length actin mRNA was translated for only 2 min before addition of the inhibitors of translation. Analysis of the reaction products over the course of the next 16 min via native-PAGE revealed that the formation of actin species I preceded that of species II, the actin–CCT complex . If the actin chains in species I were engaged with a molecular chaperone component, then an actin molecule unable to attain the native conformation might be expected to display a longer residence time with its particular chaperone. Previously, it has been shown that removal of the last 20 amino acids of yeast actin, which are essential for holding together the three noncontiguous strands of subdomain I, resulted in the loss of actin's structural integrity . To determine whether actin would accumulate in the species I complex if the acquisition of the native state was prevented, an mRNA lacking a stop codon and encoding an actin chain lacking the COOH-terminal 38 amino acids was generated and translated in vitro. In this experiment, synthesis of the NH 2 -terminal 336 amino acids will proceed (full-length actin is 374 amino acids), but in the absence of a translational stop signal the nascent chain remains bound to the ribosome. Addition of puromycin should then result in the release of nascent chains and allow for the analysis of the mass of the nascent actin chain. Translation of the truncated actin mRNA was allowed to proceed for 12 min in the presence of [ 35 S]methionine before the addition of translational initiation inhibitors. At various times thereafter, aliquots were removed, puromycin was added, ATP levels depleted via addition of apyrase, and the material analyzed by native-PAGE . Actin species I was the first and most predominant species observed. Only small amounts of the actin–CCT complex (species II) were found. Subsequent to the appearance of species I and II, low levels of the truncated actin molecule were observed to migrate near the bottom of the gel, presumably having folded into some type of monomeric-like species. Quantitation of the different actin-containing species present in Fig. 2 A revealed that the majority of the truncated actin molecule accumulated as the species I complex . Two-dimensional gel analysis (native-PAGE in the first dimension followed by SDS-PAGE in the second dimension) revealed that full-length actin could be found within all of the species . However, in the case of the 336–amino acid actin translation product, the vast majority of the truncated actin chains was present within species I. Here, very little of the truncated actin chains were found present within the CCT containing complex, species II. A heteromeric chaperone termed PFD has been described that can form a complex with chaotrope denatured full-length actin, and then deliver this target to CCT where folding to the native state commences . To see whether species I might represent actin bound to PFD, an antibody to the bacterially expressed human PFD 6 was prepared and characterized. When an immunoblot of HeLa cell and rabbit reticulocyte lysate proteins was probed with the anti–PFD 6 antibody, a single polypeptide of the appropriate size for the PFD 6 species was detected . We compared the native-PAGE migration of purified PFD with that of actin species I and II produced during the in vitro translation reaction. Following native-PAGE, the proteins were transferred to nitrocellulose and probed with the anti–PFD 6 antibody. After the enhanced chemiluminescence (ECL) image was developed, the nitrocellulose was extensively washed (to remove the ECL reagents), dried, and then reexposed to film to detect the [ 35 S]methionine-labeled actin species. As shown in Fig. 3 B, the PFD complex recognized by the antibody (lane 1) migrated in a manner similar to that observed for radiolabeled actin species I (lane 2). These observations are consistent with the idea, but do not prove, that species I consists, at least in part, of actin bound to PFD. Gel mobility shifts and immunodepletion experiments were used to further confirm the identity of species I and II. The migration of the different actin-containing species was unaffected when the material was incubated with PBS . Incubation of this material with preimmune rabbit serum affected only the migration of native monomeric actin , likely due to the presence of the actin-binding proteins gelsolin and vitamin D–binding protein within the crude rabbit polyclonal serum . Presumably the interaction of full-length actin with these two different serum proteins accounts for the doublet observed. When the anti–PFD 6 rabbit antiserum was added to the translation reaction the full-length monomeric actin species was gel shifted (due to the presence of the actin-binding proteins gelsolin and vitamin D–binding protein). Note, however, that the species I band now also disappeared upon addition of anti-PFD serum . Thus, species I contains actin and PFD. If the PFD 6 antiserum was first preincubated with purified native actin, the mobility of the radiolabeled native actin monomer was no longer altered, yet species I still disappeared . Thus, addition of native actin was sufficient to titrate all of the native actin-binding proteins present within the crude rabbit sera. When a purified mouse mAb to the CCT α subunit (antibody 23C) was added, the large (∼800-kD) species II complex disappeared . Finally, the radiolabeled actin monomer produced in these reactions was observed to gel shift upon addition of DNase I, demonstrating that this actin species had indeed reached the native state . Analysis of the 336–amino acid actin translation products again revealed that the actin species I shifted its gel mobility only upon addition of the PFD 6 rabbit serum . In addition, the antibody to CCT resulted in the disappearance of the larger actin species II . Note that the addition of either preimmune serum or DNase I now had no effect on the position of any of the species in the gel, consistent with there being no properly folded full-length actin present. We confirmed the results of the mobility shift experiments using the different antibodies or DNase first immobilized on Sepharose beads. For example, incubation of the translation reactions with immobilized anti-PFD serum again resulted in the disappearance of the actin I species . Analysis of the material bound to the immobilized PFD antiserum by SDS-PAGE confirmed the presence of radiolabeled actin . Immobilized antibodies to CCT effectively resulted in the disappearance of actin species II while the immobilized DNase effectively removed all monomeric actin . In each case the immobilized CCT antibody or DNase beads now contained significant amounts of the radiolabeled actin species . Very similar results were obtained when the immunodepletion analyses were performed on the 336 actin translation product. Again, immobilized antibodies to PFD and CCT resulted in the specific depletion of species I and II, respectively . Note that none of the 336–amino actin translation product was captured by the immobilized DNase, indicative that this actin truncation product is unable to adopt its native conformation . Interestingly, the ∼500-kD complex we termed species IV, which only formed when full-length actin mRNA was translated , was also somewhat depleted upon incubation with the anti-CCT antibody . Actin bound to this smaller complex containing at least one of the subunits of CCT appears to be native or native-like, since the immobilized DNase I also resulted in the disappearance of actin species IV . Further work will be necessary to characterize this species, as well as the species we have labeled species III. Taken together, these observations demonstrate that species I consists of actin bound to PFD, while actin species II represents actin bound to the CCT. Experiments were designed to determine whether there was a minimum length of the nascent actin chain required to interact with PFD. A series of shorter actin mRNA truncations was translated in vitro, and the products analyzed by native- and denaturing-PAGE. Time points were taken early after the addition of inhibitors of translational initiation, or at a later time when translational elongation was complete . In each case, the reactions were divided: to one half puromycin was added to release nascent chains from the ribosome, while to the other half SDS-containing buffer was added to strip the nascent chains off the ribosome . Analysis of the reaction products by SDS-PAGE revealed relatively homogeneous populations of those actin nascent chains containing ≤156 amino acids . In contrast, those reactions programmed with mRNAs encoding longer actin molecules produced significant heterogeneity in the nascent chains early during translation, suggestive of ribosomal pausing during reading of the mRNA (e.g., 336–amino acid actin). Note that in those reactions where puromycin was not added, a portion of the truncated actin molecules migrated very slowly in the SDS gel . These species probably represent actin chains (∼20–25 kD) still bound covalently to tRNA (∼25 kD). Native-PAGE analysis of the reaction products obtained from the different truncated actin mRNAs revealed when the actin–PFD complex began to form . Translation of the first 99 or 123 amino acids of actin followed by release of nascent chains with puromycin yielded no detectable actin–PFD complex. Interestingly, puromycin release of the 99 amino acid truncation product resulted in the fragment migrating in the gel with an apparent mass of ∼20–25 kD, while the 123 amino acid actin translation product migrated with an apparent mass of ∼100 kD. It is unclear whether these two actin fragments are interacting with one or more unknown proteins, but gel shift experiments using antibodies to CCT or PFD had no effect on the native-PAGE migration of these species (data not shown). The actin–PFD complex was detected only after translation of the first 145 amino acids, thus defining a minimal requirement for binding of the nascent actin chain to the chaperone. All longer actin truncation products were also found preferentially bound to PFD. A fraction of the 257–amino acid truncated actin molecule apparently had released from PFD (note those species of ∼25 kD at the bottom of the native-gel). Presumably, a portion of these chains forms some native-like structure that is not recognized by either CCT or PFD present in the lysate. Relatively low amounts of the different truncated actin products were found in the ∼800-kD CCT complex (actin–CCT). The data presented to this point are consistent with the idea that PFD interacts with nascent actin after synthesis of the first 130–145 amino acids. It remained possible, however, that complex formation between actin and PFD may occur in the reticulocyte lysate only after release of the nascent actin chains from the ribosome. To determine whether PFD can bind actin cotranslationally, the 336– and 257– amino acid actin truncations were translated, cycloheximide was added to stabilize the nascent chains, ATP was depleted, and polysomes were isolated by sedimentation through sucrose. The fractions from the polysome gradient were analyzed by SDS-PAGE and native-PAGE. In addition, RNA analysis was done to ensure that we had isolated an enriched polysome population. As shown in Fig. 5 A, the majority of the polysome-bound 336 amino acid actin translation product (marked by the bracket) was found near the bottom of the gradient (i.e., in fractions 6 and 7, these being enriched in high molecular weight RNA). The absence of endogenous globin chains (asterisk) and tRNAs in these fractions indicated that contamination of the polysome fraction by cytosol was low. When released from the purified polysomes by puromycin, the 336 actin translation product again migrated in the native gel with a native molecular mass of ∼150 kD . Immunoblot analysis of the gradient fractions using anti-CCT mAbs revealed that the majority of the ribosome-bound nascent actin chains (fractions 6 and 7) was resolved from CCT (fractions 4 and 5) during the centrifugation . To verify that the 150-kD actin containing complex released from the isolated ribosomes contained PFD, an immunodepletion experiment was performed. Incubation of the puromycin-released complexes with immobilized anti– PFD 6 resulted in the complete depletion of the truncated actin chains as shown by native-PAGE analysis . Consistent with this observation was the capture of the labeled actin by the PFD antibody beads as determined by SDS-PAGE . Note that the immobilized preimmune antibodies or DNase I slightly reduced the amount of species I . This likely is due to nonspecific binding, since the beads did not retain the radiolabeled actin chain after extensive washing . Similar results were obtained with the 257–amino acid actin truncation product . Specifically, the nascent actin chains, upon their puromycin-mediated release from the polysomes, migrated in the native gel with an apparent mass of 150 kD. Again, this material could effectively be removed by preincubation with the PFD antibody, but not with antibodies specific for CCT (data not shown). We conclude that PFD interacts with nascent actin as the actin polypeptide is being synthesized on the ribosome. Previous work has shown that nascent chains bound to hsp70 are inaccessible to low levels of added protease unless the hsp70–nascent chain complex is first dissociated by incubation with ATP. In the absence of ATP, nascent chains are sequestered from the bulk solution . We found that the actin–PFD complex, present within the whole rabbit reticulocyte lysate, was insensitive to added ATP (data not shown). Moreover, protease protection experiments revealed that little or no protection was provided to nascent actin chains bound to PFD . For example, under conditions where full-length actin exhibited relative resistance to added trypsin or chymotrypsin, the 336–amino acid translation product was rapidly and completely digested . In a second experiment, labeled full-length actin was denatured in 8 M urea, the target protein presented to purified PFD by sudden dilution, and the actin–PFD complex then isolated by gel filtration. The isolated actin–PFD complex, as well as native actin, was subjected to protease K treatment for varying times and the reaction products analyzed by SDS-PAGE . The full-length actin molecules were only partially cleaved by the added protease. In contrast, actin bound to PFD was highly susceptible to the added protease. These results, together with our observation that actin–PFD complexes are unable to bind to DNase I, indicate that nascent actin chains bound to PFD are in a relatively solvent exposed, nonnative state. To determine whether nascent chains in general interact with PFD, we translated mRNAs encoding a number of other polypeptides and looked for their possible interaction with PFD. We first examined newly synthesized α- and β-tubulin because, like actin, their productive folding has been shown to be assisted by CCT . In addition, urea-unfolded α- and β-tubulin have been shown to be targets for PFD . Finally, the yeast homologue of PFD, GimC, promotes the formation of functional α- and γ-tubulin in yeast . Full-length β-tubulin mRNA was translated in reticulocyte lysate for 8 min, inhibitors of translational initiation were added, and the reactions were further incubated to allow for polypeptide chain elongation and completion. At various times, aliquots were removed and incubated with apyrase to deplete ATP, and with puromycin to release any unfinished chains. Analysis of the reaction products by SDS-PAGE revealed that little or none of the tubulin chains had completed their synthesis after 9 min of translation, that a small amount of full-length tubulin was generated by 12 min, and that the level of full-length molecules remained relatively unchanged after 18–21 min . When analyzed by native-PAGE, two prominent tubulin containing intermediates were observed, one of ∼150 kD and the other of 800 kD . Species I, which comigrated with the actin–PFD complex (data not shown), preceded the appearance of species II . The composition of the different β-tubulin intermediates was determined by examining the effects of antibodies specific for PFD and CCT upon the native-PAGE mobility of the complexes. When anti-PFD antiserum was incubated with the translation products, only the electrophoretic mobility of species I was affected, thus it contained PFD . Species II represented full-length β-tubulin bound to CCT because the anti–TCP-1α antibody specifically altered its migration . The species migrating slightly slower than the PFD-containing complex probably represents β-tubulin complexed with one of the several tubulin-specific chaperones that facilitate tubulin heterodimer formation , while that species migrating near the bottom of the gel (indicated as tubulin) is probably α/β-tubulin heterodimer factor A–β-tubulin complex, or a mixture of the two . Analysis of the process of α-tubulin maturation in reticulocyte lysate by the same methods yielded similar results: nascent α-tubulin was observed to first interact with PFD and was then found bound to CCT. The mobility of the α-tubulin complexes formed was also specifically altered by anti-PFD and anti-CCT antibodies (data not shown). We conclude that, like actin, both α- and β-tubulin interact with PFD before delivery to the CCT. Neither PFD nor the CCT appeared to participate in the maturation pathway of four other proteins: cytosolic yeast invertase, green fluorescent protein, chloramphenicol acetyltransferase, or luciferase. A careful kinetic analysis of the different translation products via native-PAGE did not reveal any distinct intermediates of native mass similar to PFD or CCT during the maturation process (data not shown). Based on these observations we conclude that unlike actins or tubulins, maturation of these four proteins does not involve PFD or CCT. Here we have shown that the cytoskeletal proteins, actin, and α- and β-tubulin undergo a defined series of interactions with at least two chaperones during the course of maturation. In the case of β-actin, cotranslational interaction of the nascent actin chain with PFD occurs following synthesis of the first 145 amino acids. Once engaged, the actin–PFD complex persists until the completion of actin synthesis. Actin molecules bound to PFD are nonnative and accessible to added proteases. Upon completion of synthesis, full-length actin molecules are transferred to CCT where folding to the native state is thought to occur . A similar maturation pathway applies in the case of both α- and β-tubulin, although interaction of nascent β-tubulin with PFD occurs later than that observed for actin (i.e., after ∼250 amino acids have been synthesized; data not shown). Consequently, as in the case of actin, we suspect that once bound to the nascent chain, PFD remains engaged until the completion of tubulin synthesis, probably until transfer to CCT has occurred. Support for the aforementioned pathway of actin and tubulin maturation follows from a number of observations. First, kinetic analysis of translation products derived from full-length actin shows that the newly synthesized protein first accumulated in a complex with PFD. Over time the newly synthesized actin molecules were then found in a complex with CCT, before their appearance as monomeric actin . Only the monomeric actin appeared properly folded as evidenced by its ability to interact with DNase I . Second, translation of a truncated actin mRNA resulted in the majority of the puromycin-released actin becoming complexed with PFD; very little of the truncated actin molecules was found in the chaperonin complex . To rule out the possibility that the truncated actin chains released prematurely from the ribosome were simply interacting with PFD molecules present within the reticulocyte lysate, polysomes containing nascent actin were isolated. Subsequent release of the nascent chains and analysis by native-PAGE revealed actin in a complex with PFD, but not with the CCT . The most likely interpretation of these data is that PFD was associated with polysomes based on its interaction with nascent actin chains. Analysis of the various actin intermediates by native-PAGE followed by SDS-PAGE demonstrates that nascent, as well as full-length, actin could be found complexed with PFD. Relatively low levels of the puromycin-released nascent actin chains could be found in a complex with CCT: rather, the full-length actin molecules appeared to be the preferential binding partner for CCT. Similar results were found for α- and β-tubulin . Taken together, our results lead us to conclude that during the course of their maturation, these cytoskeletal proteins interact first with PFD and, upon completion of their synthesis, are transferred over to the chaperonin complex for their folding to the native state. We cannot rule out that other pathways might also feature in the maturation of these proteins. Frydman and Hartl described the cotranslational interaction of nascent actin with the hsp70 chaperone, as well as with CCT . While we also observed an interaction of newly synthesized actin with CCT, this interaction appeared to occur primarily after release of the actin chain from the ribosome. It remains possible that in the absence of PFD actin and tubulin maturation may still occur, but presumably with slower kinetics and reduced fidelity. Indeed, this is precisely the case in yeast strains carrying mutations in the different PFD subunits . Through binding cotranslationally to its target proteins, which are in a relatively unfolded state, PFD may prevent misfolding/aggregation during synthesis or after release from the ribosome. In this context, binding by PFD may be a prerequisite for efficient substrate presentation to the CCT. We cannot rule out the possibility that PFD also participates in the rebinding of actin or tubulin molecules that are released from the CCT in a nonnative state. Recently, it has been reported that the yeast PFD homologue, GimC, acts primarily in a complex with the CCT during the folding of actin and perhaps also after release of actin from the chaperonin . Our studies add to this complex picture by showing that PFD (and presumably GimC) act, at least in part, by binding cotranslationally to the actin chain and subsequently presenting the full-length actin (and tubulin) polypeptide to the chaperonin complex. In vivo, there is a five- to eightfold decrease in the rate of actin folding in GimC (PFD) knockout strains of yeast . The authors suggest that GimC (PFD) prevents the release of nonnative actin folding intermediates from the chaperonin, and thereby accelerate their folding. Alternatively, it has been suggested that if the actin released from CCT has not yet reached the final native state, then the interaction of actin with PFD (GimC) ensures that the substrate will be retargeted to CCT . The data presented here may help to explain the slower appearance of folded actin observed in yeast GimC mutants : the absence of a functional GimC (PFD) chaperone may reduce the efficiency of transfer of the newly synthesized actin chain from the ribosome to CCT. Exactly what property (sequence) of nascent actin or tubulin dictates their interaction with PFD remains to be determined. Interestingly, several amino acids (present within amino acids 103–133 of actin), which have emerged from the ribosome when actin first binds to PFD, are destined to pack into the native actin structure against residues found in the extreme COOH terminus . Perhaps PFD helps to prevent misfolding of residues 103–133 until the COOH-terminal region of actin has been synthesized. Finally, it is important to note that so far we have been unable to demonstrate any significant interaction of other nascent/newly synthesized proteins with PFD or CCT. Hence, we suspect that like the chaperonin with which it interacts , PFD appears to have a relatively narrow range of target proteins. However, the family of proteins that possess the actin fold (i.e., potential PFD substrates) is growing. Members of the actin-related protein family function in diverse essential processes, such as transcriptional regulation , orientation of the mitotic spindle, nuclear migration , and membrane polarity and endocytosis . Therefore, it will be interesting to see whether these various relatives of actin similarly require the PFD–CCT pathway for their proper maturation. | Study | biomedical | en | 0.999998 |
10209024 | Polyclonal antibody against full-length, recombinant CALNUC was generated and affinity purified as previously described . Polyclonal anti–α-mannosidase II (Man II) was prepared as described . Monoclonal anti–Man II (53FC3) and polyclonal antibody against denatured Man II were gifts from Drs. B. Burke (University of Alberta, Alberta, Canada) and K. Moremen (University of Georgia, Athens, GA), respectively. Monoclonal anti–mouse IP 3 R-1 (18A10) was kindly provided by Drs. A. Miyawaki and K. Mikoshiba (University of Tokyo, Tokyo, Japan) . Polyclonal antibody against calnexin was a gift from Dr. J.J.M. Bergeron (McGill University, Montreal, Canada). Cross-absorbed Texas red–conjugated donkey anti–rabbit F(ab′) 2 was obtained from Jackson ImmunoResearch Laboratories, and affinity-purified goat anti–rabbit IgG (H+L) conjugated to HRP was from Bio-Rad. 45 CaCl 2 was obtained from NEN Life Science Products. Supersignal chemiluminescent reagent was purchased from Pierce. All chemical reagents were from Sigma Chemical Co. except as indicated. Full-length CALNUC cDNA was amplified by PCR using 5′-CGCGCGGCAGCCATATGCCTACCTCTGTG-3′ and 5′-CGGAATTCGGATCCTTATAAATGCTGAGAATC-3′ as primers. PCR was carried out using 100 pmol of each primer, 2 ng CALNUC cDNA, 200 μM dNTP, 2.5 U PFU polymerase (Stratagene), and PCR reaction buffer in a total volume of 50 μl. PCR products were purified using a QIAquick PCR Purification kit (Qiagen) and subcloned into the pET-28a(+) vector (Novagen) at BamHI/NdeI restriction sites, followed by transformation into Escherichia coli BL21(DE3). Expression of CALNUC protein was induced with 1 mM isopropyl β- d -thiogalactoside (IPTG) ( Pharmacia Biotech ) at 18°C for 4 h at a bacterial density of OD 600 ≥ 1.0. To purify His6-CALNUC protein, transformed E . coli were suspended in binding buffer containing 20 mM sodium phosphate and 500 mM NaCl (pH 7.5), and sonicated using a Microson Ultrasonic Cell Disrupter (Heat Systems). Lysates were incubated with Ni-NTA agarose (Qiagen), washed with 20 mM sodium phosphate, 500 mM NaCl at decreasing pH (8.0, 6.0, and 5.3), and bound proteins were sequentially eluted with an imidazole step gradient (10 mM to 1 M). Fractions containing a single band of purified CALNUC detected by silver staining were pooled and dialyzed against TBS containing 150 mM NaCl and 10 mM Tris-HCl (pH 7.4) at 4°C, and subsequently concentrated using an Ultrafree-15 (Biomax-50K) filter ( Millipore ). Highly purified His6-CALNUC [0.6 mg/liter of transformed BL21(DE3)] was obtained. Equilibrium dialysis was performed essentially as previously published . Ca 2+ -free solution was prepared by treatment of deionized water with a UniPure I Water Purification System (Solution Consultants) and Chelex 100 ion exchange resin (Bio-Rad) . Equilibrium dialysis was performed using a Dialysis System ( GIBCO BRL ). 0.25 mg Ca 2+ -depleted CALNUC was incubated with 0.35 μCi/ml 45 CaCl 2 and different concentrations of cold Ca 2+ at 4°C for 16 h, followed by assessment of radioactivity using a LS 6000IC Liquid Scintillation System ( Beckman Instruments ) in EcoLume liquid scintillation cocktail (ICN). Scatchard analysis was performed using CA Cricket Graph III software (Computer Associates International). Amino acid sequences of CALNUC, Cab45 , and calmodulin (CaM) were obtained through Entrez on the National Center for Biotechnology Information's (NCBI) World Wide Web home page. Alignment of EF-hand motifs was performed using MacVector 6.0 software (Oxford Molecular Groups-IBI). HeLa cells were maintained in DME high glucose medium (Irvine Scientific) supplemented with 10% FCS (Life Technologies Inc.). Cells were used as 80% confluent monolayers for transfection. Transfected EcR-CHO cells were cultured in Ham's F12 medium (CORE Cell Culture Facility, University of California, San Diego, CA) with 10% FCS (Life Technologies), 250 μg/ml Zeocin (Invitrogen), and 750 μg/ml G418 sulfate ( Calbiochem ). All media contained 100 U/ml of penicillin G and 100 μg/ ml of streptomycin sulfate. NRK cells were cultured as previously described . CALNUC cDNA was amplified by PCR with the primers 5′-CGCGGATCCATGCCTACCTCTGTG-3′ and 5′-CCATGCCATGGCTAAATGCTGAGAATCC-3′. GFP cDNA was also amplified by PCR with the primers 5′-TCATGCCATGGTGAGCAAGGG-3′ and 5′-ATAGTTTAGCGGCCGCTTACTTGTACAGCTC-3′. PCR products were purified and digested, respectively, with BamHI, NcoI, and NotI ( New England Biolabs ). CALNUC and GFP cDNA were subcloned into the pcDNA3 vector (Invitrogen) by three-fragment ligation to obtain a CALNUC-GFP/pcDNA3 construct with GFP ligated to the 3′ (COOH terminus) of CALNUC. CALNUC(ΔEF-1), in which the α helix (Asp 227 –Leu 239 ) of the first EF-hand (EF-1) domain was deleted, was obtained by PCR with the primers 5′-CGCGGATCCATGCCTACCTCTGTG-3′/5′-CCCAAGCTTATGCAGTATGAAGAA-3′, and 5′-CCCAAGCTTGAAGCTCTGTTTACC-3′ / 5 ′ -CCATGCCATGGCTAAATGCTGAGAATCC-3 ′ . CALNUC(ΔEFs-1,2), in which both EF-1 and EF-2 domains (Asp 227 – Phe 291 ) were deleted, was prepared with primers of 5′-CGCGGATCCATGCC TACC TCTGTG- 3 ′ / 5 ′ -CCCAAGCT TATGCAGTATGAAGAA-3′, and 5′-CCCAAGCTTCTGGCATCCACACAG-3′/5′-CCA- TGCCATGGCTAAATGCTGAGAATCC-3′. CALNUC mutants and the GFP tag were subcloned into the pcDNA3 vector by four-fragment ligation with HindIII and NcoI as internal restriction linker sites. Fidelity of the constructs was verified by automated DNA sequencing (CFAR, University of California, San Diego, CA). cDNA constructs were transformed into E . coli DH5α, followed by extraction and purification using QIAGEN Plasmid Midi/Mega Kits (Qiagen) and UltraPure CsCl (optical grade) ( GIBCO BRL ). To express wild-type or truncated CALNUC-GFP in HeLa cells, 1 μg purified DNA was transfected into HeLa cells (33-mm dish, 80% confluence) using 6 μg lipofectamine ( GIBCO BRL ). Transfected cells were grown in serum and antibiotic-free high glucose DME medium for 5 h followed by replacement with regular culture medium. GFP cDNA amplified by PCR with the primers 5′-TCGCGGATCCATGGTGAGCAAGGG-3′ and 5′-ATAGTTTAGCGGCCGCTTACTTGTACAGCTC-3′ was subcloned into the pcDNA3 vector at BamHI/ NotI restriction sites, followed by transfection into HeLa cells as described above and G418 selection (0.75 mg/ml) for 4 d. Cells expressing GFP were sorted by flow cytometry (Ex/Em: 488/530 ± 15) (FACStar Plus ® ; Becton Dickinson ) in the UCSD Flow Cytometry Core Facility. The top 0.12% of the positive cells was collected and maintained in media containing 0.75 mg/ml G418 until confluent. Selection by sorting was repeated three times until 100% of the cells expressed GFP (data not shown). CALNUC cDNA was amplified by PCR and subcloned into the pIND vector (Invitrogen) at BamHI/NotI restriction sites. EcR-CHO cells (Invitrogen) stably expressing the ecdysone receptor (RxR and VgEcR) were transfected with CALNUC/pIND plasmid DNA using lipofectamine as described above followed by selection for G418 resistance (0.4 mg/ml) for 18 d. Cells were split into 96-well plates by serial dilution, 0.5 cells/well, and subsequently reselected with G418 (0.75 mg/ml). Four clones overexpressing CALNUC after induction with muristerone A (Invitrogen) were obtained; one of these, EcR-CHO-CALNUC-1 (CPC-22A), was used for these experiments. CALNUC-GFP was directly visualized using a Zeiss Axiophot microscope and an FITC-filter (Ex/Em: 485/510). For immunofluorescence, cells on coverslips were fixed with 2% paraformaldehyde (50 min), permeabilized with 0.1% Triton X-100 (10 min), and incubated with affinity-purified anti-CALNUC IgG (6 μg/ml), anti–Man II serum (1:300), or anticalnexin serum (1:100) as previously described . Detection was with Texas red– or FITC-conjugated donkey anti–rabbit F(ab′) 2 . In some cases cells were doubly stained for CALNUC and either a mouse mAb against Man II (40 μg/ml) or the IP 3 R-1 (1.25 μg/ml) and appropriate secondary antibodies. Specimens were examined with either a Zeiss Axiophot equipped for epifluorescence or a Bio-Rad confocal microscopy equipped with Lasersharp 3.1 software (Bio-Rad) and a krypton-argon laser. Images were processed with Scion Image and Adobe Photoshop (Adobe Systems) software. Sucrose gradient flotation of Golgi fractions was carried out using a protocol similar to those previously published with minor modifications. In brief, microsomal membranes were resuspended in 1.5 ml 55% sucrose (wt/wt), loaded at the bottom of a sucrose step gradient consisting of 40, 35, 30, 25, and 20% (wt/wt in 1 mM Tris-HCl, pH 7.5), and centrifuged at 85,500 g for 16 h at 4°C using a SW-40Ti rotor (Beckman). 20 fractions were collected from the bottom, followed by SDS-PAGE and immunoblotting for calnexin (an ER marker), Man II (a Golgi marker), and CALNUC. Rat liver Golgi fractions, membrane (100,000 g pellet) and cytosolic (100,000 g supernatant) fractions were prepared from postnuclear supernatants of transfected HeLa or EcR-CHO-CALNUC cells as previously described . Proteins were separated by 5 or 10% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with affinity-purified anti-CALNUC IgG, anticalnexin, and anti–Man II serum followed by HRP-conjugated anti–rabbit IgG and detection by ECL . The procedures followed were those reported previously . Cells (2 × 10 6 ) transfected with CALNUC-GFP or GFP alone were incubated with 45 Ca 2+ (2 μCi/ml) for 48 h to reach 45 Ca 2+ equilibrium after which they were washed three times in Krebs-Ringer-Hepes (KRH) buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 2 mM CaCl 2 , 6 mM glucose, and 25 mM Hepes, pH 7.4) and five times with PBS. 45 Ca 2+ was extracted with 0.1 N HCl (30 min at room temperature) and radioactivity assessed as described above. To examine 45 Ca 2+ release after stimulation, washed cells were resuspended in KRH supplemented with 3 mM EGTA and stimulated at room temperature with 100 μM ATP or sequentially stimulated with 0.1 μM Tg, 2 μM ionomycin, and 2 μM monensin, 5 min each. Equal aliquots (10 6 cells) were collected after each stimulation, followed by centrifugation at 14,000 rpm (30 s) and quantification of 45 Ca 2+ in the supernatant. Noninduced or induced EcR-CHO-CALNUC cells were loaded with 1 mM Fura-2 AM (Molecular Probes Inc.) in Ham's F12 medium/0.5% FCS at 22°C for 1 h, washed with Ca 2+ -free HBSS (Irvine Scientific) followed by addition of 100 μM ATP. Ca 2+ release was monitored by Ca 2+ imaging performed on a Zeiss Axiovert microscope equipped with a cooled charge-coupled CCD camera (Photometrics) and MetaFluor software ( Universal Imaging ). Dual-excitation ratio imaging was obtained using two excitation filters (340DF20 and 380DF20) (Omega Optical and Chroma Technology) mounted on a filter wheel (Lambda 10-2; Sutter Instruments ), a 420DRLP dichroic mirror, and a 510DF80 emission filter. The procedures used were basically similar to those published with minor modifications. To examine equilibrium 45 Ca 2+ uptake, EcR-CHO-CALNUC cells induced with 5 μM ponasterone A for 24 h in a 6-well culture plate (10 6 cells/well) were permeabilized at 20°C for 4 min with saponin (50 μg/ml) in loading buffer (140 mM KCl, 20 mM NaCl, 2 mM MgCl 2 , 2 mM ATP, 0.1 mM EGTA, 20 mM Pipes, pH 6.80), and 0.13 μM free Ca 2+ calculated for conditions of pH 6.80, at 20°C . Cells were washed four times with loading buffer and subsequently loaded with 45 Ca 2+ (10 μCi/ml) for various times (10–60 min). They were then rinsed five times with loading buffer (30 s), stored 45 Ca 2+ was extracted with 1 ml 0.1 N HCl for 30 min, and 0.5-ml aliquots were counted. To investigate 45 Ca 2+ mobilization by IP 3 , induced and permeabilized EcR-CHO-CALNUC cells were loaded with 10 μCi/ml of 45 Ca 2+ as above for 45 min. After washing (five times over 1–1.5 min), cells were challenged with 10 μM IP 3 ( d -myo-inositol 1,4,5-trisphosphate potassium salt) in loading buffer, 1 ml/well. Solutions were collected at 2-min intervals, replaced with loading buffer containing IP 3 , and counted. To quantify endogenous CALNUC in rat liver Golgi fractions, a linear standard curve was obtained for purified His6-CALNUC (1.3–40 ng) by immunoblotting and densitometric analysis (data not shown). Endogenous CALNUC was found to be present in pooled Golgi light and heavy fractions from rat liver at a concentration of 3.8 μg/mg Golgi protein, i.e., ∼0.4% of the total Golgi protein (includes both Golgi resident proteins and cargo in transit through the Golgi). NRK cells were found to have 0.02 μg CALNUC/ 10 6 cells, or 2.5 × 10 5 CALNUC molecules/NRK cell. An ideal EF-hand Ca 2+ -binding motif has an α helix– loop–α helix structure in which oxygen ligands (O) provided by carboxy side chains of Asp (D)/Glu (E), carbonyl groups (C′O) of the peptide main chain and H 2 O constitute the Ca 2+ -binding site, and a hydrophobic amino acid (φ) and a Gly (G) are essential for Ca 2+ binding . CALNUC and Cab45 are the only EF-hand, Ca 2+ -binding proteins identified so far in the Golgi. Since their Ca 2+ -binding constants are not yet known, in order to predict and compare the Ca 2+ -binding properties of these two proteins, we compared the EF-hand primary structures of CALNUC (two EF-hands), Cab45 (six EF-hands), and CaM (four EF-hands). Cab45's and CaM's EF-hand motifs are similar , and CaM's Ca 2+ -binding properties have been well characterized. As shown in Fig. 1 A, CALNUC EF-1, Cab45 EF-2 and -5, and all four CaM EF-hand structures constitute ideal EF-hand motifs. CALNUC EF-1 and EF-2 are strikingly similar to CaM EF-4 and Cab45 EF-5, but CALNUC EF-2 has an Arg (R) instead of Gly at residue 6 . This suggests that CALNUC has only a single ideal EF-hand motif, EF-1. To investigate the binding affinity of CALNUC for Ca 2+ , we performed equilibrium dialysis. Purified recombinant His6-CALNUC was used based on a report that recombinant calreticulin (CRT) was comparable to native CRT in its Ca 2+ -binding capability . Scatchard analysis of the binding curve indicates that CALNUC binds Ca 2+ with a high affinity binding constant ( K d = 6.6 μM) and a low capacity, ∼1.1 μmol Ca 2+ /μmol protein, suggesting only one high affinity, low capacity Ca 2+ -binding site on CALNUC. To further investigate Ca 2+ binding to CALNUC in vivo, we expressed CALNUC-GFP by transient transfection in HeLa cells and generated an inducible cell line, EcR-CHO-CALNUC, stably expressing CALNUC. By immunoblotting, CALNUC-GFP (90 kD) was detected in transiently transfected HeLa cells but not in nontransfected cells . The majority of the CALNUC (∼85%) was associated with membranes (100,000 g pellet) and the remainder (15%) was present in the cytosolic fraction (100,000 g supernatant). Three additional bands , also visualized after in vitro translation (data not shown), were also seen. They could be products of protein degradation or mistranslated CALNUC retained in the cytosol. By immunofluorescence the distribution of CALNUC-GFP overlapped with that of the Golgi marker Man II , indicating that the majority of the CALNUC-GFP is correctly targeted to the Golgi. In EcR-CHO-CALNUC cells induced with muristerone A or ponasterone A (0.1–10 μM) for 24 h, we found a linear increase in the expression of CALNUC with increasing amounts of added hormone . The ratio of CALNUC in membrane versus cytosolic fractions was similar to that of CALNUC-GFP in HeLa cells (data not shown). By immunofluorescence the distribution of CALNUC again overlapped with that of Man II in the Golgi region in EcR-CHO-CALNUC cells induced with 10 μM muristerone A for 24 h, and was distinct from that of the ER marker, calnexin . Next we analyzed the distribution of CALNUC in induced EcR-CHO-CALNUC cells using an established procedure for flotation of Golgi membranes and their separation from ER membranes. As shown in Fig. 4 , we found that CALNUC and Man II cosedimented and peaked in fractions 12–15 with sucrose densities similar to those previously reported (1.10–1.14 g/ml) for CHO cells. By contrast, the ER marker, calnexin, peaked in denser fractions 7–11 (1.16–1.19 g/ml). These results together with the immunofluorescence findings demonstrate that overexpressed CALNUC is found in the Golgi and is consistent with our previous conclusion that overexpression does not lead to mistargeting of CALNUC. To assess whether overexpressed CALNUC-GFP binds Ca 2+ in the Golgi, we carried out in vivo equilibrium Ca 2+ uptake. The 45 Ca 2+ loading time was ∼48 h, the time shown previously to be long enough to reach 45 Ca 2+ equilibrium in cultured cells . 45 Ca 2+ uptake by HeLa cells transiently overexpressing CALNUC-GFP was 2.5-fold that of nontransfected HeLa cells or those stably expressing GFP alone . Similarly, there was a threefold increase in 45 Ca 2+ taken up by induced (5 μM muristerone A for 48 h) versus noninduced EcR-CHO-CALNUC cells . These results demonstrate that Golgi-associated CALNUC binds Ca 2+ in vivo and most likely is responsible for sequestering Ca 2+ in the Golgi lumen. To investigate whether EF-1 is indeed the sole Ca 2+ -binding motif in CALNUC, we examined Ca 2+ binding in HeLa cells transiently transfected with truncated CALNUC-GFP mutants. When the α helix of EF-1 (Asp 227 – Leu 239 ) or both EF-1 and EF-2 (Asp 227 –Phe 291 ) were deleted from CALNUC, its Ca 2+ -binding capability was completely abolished . Mistargeting could be ruled out since the majority of the mutant CALNUC-GFP was detected in the Golgi region by fluorescence. The results obtained from this in vivo Ca 2+ -binding analysis provide direct evidence that CALNUC binds Ca 2+ in the Golgi, and EF-1 constitutes the sole Ca 2+ -binding site on CALNUC. The latter is in agreement with the data shown in Fig. 1 . To further investigate the characteristics of the Golgi Ca 2+ pool, we performed experiments similar to those done previously to characterize the ER Ca 2+ pool in cells overexpressing CRT . When HeLa cells transiently overexpressing CALNUC-GFP or EcR-CHO-CALNUC cells stably expressing CALNUC were treated with the SERCA inhibitor Tg , ∼73% and 70%, respectively, of the 45 Ca 2+ was released , suggesting the existence of SERCA on Golgi membranes. Since some Tg-insensitive organelles are capable of retaining Ca 2+ after Tg treatment, we subsequently treated cells with the Ca 2+ ionophore ionomycin to release the remaining stored 45 Ca 2+ . Nearly all the remaining 45 Ca 2+ (∼20–25%) was released by ionomycin . In view of the fact that ionomycin is inactivated in acidic compartments such as secretory granules and endosomes, we further treated cells with monensin, a carboxylic sodium proton ionophore which releases Ca 2+ from acidic compartments and found <5% of the 45 Ca 2+ was released. Cells overexpressing CALNUC-GFP or induced EcR-CHO-CALNUC cells released twice as much 45 Ca 2+ as nontransfected HeLa cells, HeLa cells stably expressing GFP alone, or noninduced EcR-CHO-CALNUC cells. The fact that the majority of the 45 Ca 2+ taken up by CALNUC was released by Tg suggests that both CALNUC and SERCA play a key role in sequestering 45 Ca 2+ in the Golgi, a conclusion in agreement with the recent description of SERCA associated with isolated Golgi fractions . We next examined whether or not Ca 2+ sequestered in the Golgi is released after agonist challenge. Extracellular ATP is known to activate phospholipase C (PLC) via binding to G protein–coupled nucleotide receptors on the cell surface . Activated PLC promotes production of IP 3 which binds to IP 3 R on the ER and triggers Ca 2+ mobilization. To investigate whether Ca 2+ sequestered by overexpressed CALNUC in the Golgi could be released by agonist, we examined Ca 2+ release in EcR-CHO-CALNUC cells by Ca 2+ imaging after ATP challenge. The results demonstrated that the ratio, 340:380, was doubled in cells induced with 2.5 μM ponasterone A for 24 h compared with noninduced cells, suggesting that more Ca 2+ was released from induced cells. Similar results were also obtained when induced EcR-CHO-CALNUC cells were loaded with 45 Ca 2+ . These results obtained by two different methods suggest that the Golgi Ca 2+ store is sensitive to IP 3 generated after ATP binding. To obtain direct evidence that IP 3 is able to release Ca 2+ from the Golgi, 45 Ca 2+ uptake and release studies were performed on permeabilized EcR-CHO-CALNUC cells. Fig. 8 A reveals that 45 Ca 2+ is rapidly taken up by both induced and noninduced permeabilized cells, but approximately twice the amount of 45 Ca 2+ was sequestered by cells overexpressing CALNUC. Steady state was achieved 45 min after loading, which was slower than reported for Swiss 3T3 cells (20 min) . 45 Ca 2+ release was then stimulated by addition of IP 3 . The ratio of 45 Ca 2+ released from induced versus noninduced cells was ∼2:1. These results support the previous report of Pinton and colleagues suggesting that both Golgi membranes and ER membranes bear IP 3 R. In view of the functional evidence for the existence of IP 3 R on the Golgi, we carried out immunofluorescence studies on NRK cells and induced EcR-CHO-CALNUC cells using a mAb that recognizes IP 3 R-1. IP 3 R-1 was found throughout the cytoplasm and concentrated in the Golgi region which is compatible with both an ER and Golgi localization. Confocal analysis showed that the distribution of IP 3 R-1 overlaps with that of CALNUC in the juxtanuclear region, suggesting that IP 3 R-1 and CALNUC colocalize on Golgi membranes. As mentioned by Pinton and co-workers , it was not possible to carry out reproducible immunogold localization by immunoelectron microscopy with the antibody available. The Golgi complex has been recently identified as a Ca 2+ -enriched compartment whose total Ca 2+ concentration is >0.1 mM , but the question of how Ca 2+ is sequestered in the Golgi has remained unanswered. Previously we showed that CALNUC is the major Ca 2+ -binding protein in Golgi fractions from rat liver detected by 45 Ca 2+ overlay . In this study we provide evidence that CALNUC binds Ca 2+ in the Golgi in vivo, because overexpression of CALNUC in the Golgi led to a two- to threefold increase in Ca 2+ storage based on Ca 2+ equilibrium loading. This suggests that CALNUC is directly involved in maintenance of Ca 2+ storage and thereby in Ca 2+ homeostasis in the Golgi. Equilibrium dialysis demonstrated the existence of only a single high affinity ( K d = 6.6 μM)/low capacity (∼1 mol Ca 2+ /mol protein) binding site on recombinant CALNUC. CALNUC's low Ca 2+ -binding capacity in the Golgi might be compensated for by its abundance (3.8 μg/mg Golgi protein). The demonstration of a single, high affinity Ca 2+ -binding site is in keeping with the fact that CALNUC possesses two EF-hand motifs but only one, EF-1, has the structure expected for high affinity calcium binding. EF-2 has an Arg (R) instead of a Gly (G) at residue 6 of the EF-hand loop region. Arg is supposed to disrupt the EF-hand motif and abolish its Ca 2+ -binding capacity . CALNUC's EF-1 has the highest homology to the COOH-terminal EF-4 of CaM which constitutes the high affinity Ca 2+ -binding site of CaM . Moreover, the Ca 2+ -binding capability of CALNUC EF-1 was demonstrated previously by 45 Ca 2+ overlay on truncated CALNUC. When EF-2 was deleted, Ca 2+ binding was maintained, but when both EF-1 and EF-2 were deleted, Ca 2+ -binding capability was lost . In this study, we further demonstrated that truncated CALNUC with either the EF-1 α helix (Asp 227– Leu 239 ) or both EF-1 and EF-2 domains (Asp 227 –Phe 291 ) deleted lost Ca 2+ -binding capability completely. The majority of each of the CALNUC mutant proteins was still targeted to the Golgi region as monitored via the GFP tag. Collectively, these data suggest that EF-1 may constitute the sole high affinity Ca 2+ -binding site on CALNUC. Characterization of the Ca 2+ pool in HeLa and CHO cells overexpressing CALNUC provides several important new pieces of information. 45 Ca 2+ sequestered in the Golgi in cells overexpressing CALNUC was largely released by Tg, an irreversible inhibitor of the SERCA Ca 2+ pump, providing in vivo evidence for the existence of SERCAs on Golgi membranes. SERCAs were also assumed to be localized on Golgi membranes because it was shown previously that the p-type, Tg-sensitive SERCA Ca 2+ pump was essential for Ca 2+ uptake into isolated Golgi fractions in vitro . Our results also suggest that the increase in 45 Ca 2+ uptake in cells overexpressing CALNUC is not likely to be due to the presence of CALNUC in the cytosol or another recently reported Tg- and IP 3 -insensitive Ca 2+ pool since the majority of the Ca 2+ was released only after SERCA was inhibited. Our finding that only a small amount of the Ca 2+ remaining after Tg treatment was released by subsequent ionomycin treatment might be due to incomplete depletion of Ca 2+ from the Golgi by Tg, since the existence of a Tg-insensitive/ionomycin-sensitive–plasma membrane calcium ATPase Ca 2+ pump on Golgi membranes has also been reported recently . The fact that monensin treatment which depletes Ca 2+ from acidic compartments (secretory vesicles, granules, trans-Golgi network) did not release a significant amount of Ca 2+ demonstrates that Ca 2+ was not sequestered in an acidic compartment. Thus, our current results from in vivo studies suggest that the Ca 2+ -binding protein CALNUC together with SERCA Ca 2+ pumps are responsible for the maintenance of the Golgi Ca 2+ storage pool. We also investigated the agonist sensitivity of the Golgi Ca 2+ pool. It was shown recently that the Golgi Ca 2+ store is sensitive to histamine, an agonist known to be coupled to IP 3 generation , suggesting that there may be IP 3 R on Golgi membranes. Here we used extracellular ATP, another agonist known to generate IP 3 after binding to plasma membrane nucleotide receptors (P 2y -purinoceptors) , to investigate the sensitivity of the Golgi Ca 2+ store to IP 3 . ATP challenge is coupled to IP 3 production via activation of PLC , and binding of IP 3 to IP 3 R on the surface of Ca 2+ pool releases intracellular Ca 2+ . When ATP was added to induced EcR-CHO-CALNUC cells, there was a rapid release of sequestered Ca 2+ revealed by both Ca 2+ imaging and 45 Ca 2+ which far exceeded that released from noninduced cells. Moreover, IP 3 directly triggered 45 Ca 2+ mobilization from the Golgi in permeabilized EcR-CHO-CALNUC cells. Thus, our biochemical results and those of Pinton et al. using histamine as agonist suggest that the Golgi apparatus bears IP 3 R. The assumption that IP 3 R are expressed on the Golgi is supported by our immunofluorescence observations suggesting a dual localization of IP 3 R-1 on both ER and Golgi membranes. CHO cells were found previously to express ample IP 3 R-1 by immunoprecipitation using mAb 18A10 which specifically recognizes the COOH terminus of IP 3 R-1. A major controversy in the physiology of intracellular Ca 2+ stores concerns the mechanism by which their depletion triggers influx of Ca 2+ through the plasma membrane. In vertebrate cells, it has been assumed generally that the relevant store is the ER . However, because both ER and Golgi accumulate Ca 2+ via SERCAs and release Ca 2+ via IP 3 receptors, both should undergo depletion roughly in parallel, so one cannot yet exclude a role for the Golgi in controlling plasma membrane Ca 2+ influx. In yeast, store-operated Ca 2+ influx appears to be controlled mainly at the Golgi, because genetic deletion of the Golgi Ca 2+ pump encoded by PMR1 increases the influx of extracellular Ca 2+ . Therefore, we tried to distinguish between ER and Golgi contributions by testing whether overexpression of CALNUC in Xenopus oocytes affected the store-operated Ca 2+ current, Isoc . If the Golgi were important, increasing the quantity of Ca 2+ buffer in its lumen should diminish or delay Isoc . Overexpression of CALNUC (via microinjection of its mRNA) increased the 45 Ca 2+ content of oocytes analogously with Fig. 5 and appeared by fluorescence microscopy to be colocalized with the Golgi marker galactosyltransferase fused to GFP . However, CALNUC overexpression did not significantly affect Isoc, either partially activated by the membrane-permeant Ca 2+ buffer TPEN or maximally activated by the ionophore ionomycin. This negative result might seem to argue against a major role for the Golgi in controlling Ca 2+ influx into oocytes, but a firm conclusion would require additional controls such as immunoelectron microscopic localization of CALNUC and evidence that comparable increases in ER buffering do affect Isoc. Previously, we demonstrated significant homology between CALNUC and CRT and two conserved motifs, AY(I/A)EE and QRLX(Q/E)E(I/E)E, located in the C-domain of CRT (aa 337–341 and 365–372 ) . However, the homologous regions do not involve Ca 2+ -binding domains. CRT lacks EF-hand motifs but possesses a high affinity/low capacity and a low affinity ( K d = 2 mM)/ high capacity (21 μmol Ca 2+ /μmol protein) Ca 2+ -binding site constituted by clusters of ∼35 Asp (D)/Glu (E) located in CRT's C-domain. In the future it will be of interest to examine whether CALNUC can function like CRT, its ER-resident counterpart , to maintain a high Ca 2+ concentration required for Golgi functions, e.g., sorting, lectin binding, budding, and concentration of cargo into regulated secretory granules. In summary, this study demonstrates that CALNUC, an abundant Golgi resident protein and the major Golgi Ca 2+ -binding protein, together with SERCA Ca 2+ pumps and IP 3 R are involved in the maintenance of the Ca 2+ storage pool in the Golgi. Further investigation of several remaining intriguing questions including whether the binding of Ca 2+ to CALNUC regulates membrane traffic or posttranslational processing events in the Golgi, should shed light on the biological functions of CALNUC and on the Golgi Ca 2+ pool. | Study | biomedical | en | 0.999996 |
10209025 | Yeast strains used in this study are listed in Table I . Media preparations and genetic techniques were performed as described . Either glucose (D at 2%) or glycerol (G at 2%) was used as a carbon source as specified. W303 haploid cells were mutagenized using either EMS, nitrosoguanidine or UV light to 10% viability and screened for temperature sensitive growth on YPG. Cells from these colonies were grown at 37°C on YPD for ∼100 generations and were screened for loss of mtDNA using DAPI and fluorescence microscopy. Complementation analysis using a recessive truncated loss of function allele of MGM101 (strain CS6-1D, kindly provided by G.D. Clark-Walker) revealed that one mutant from this screen was allelic to MGM101 [JNY131( mgm101-2 )], The Australian National University. Sporulation and tetrad analysis of this cross confirmed the mutation was at the MGM101 locus. To characterize JNY131( mgm101-2 ), cultures of W303 or JNY131 ( mgm101-2 ) were grown at permissive temperature (22°C) to log phase in YPG, washed into YPD and were incubated at either permissive or nonpermissive temperatures. At various time intervals, aliquots were taken and cells were analyzed for respiratory competence, the presence of mtDNA and mitochondrial morphology. To analyze for respiratory competence, cells were plated onto YPD plates and colony color was assessed and quantitated. Because all strains analyzed contained the ade2 mutation, on YPD, white and sectored colonies were classified as respiratory deficient and red colonies were classified as respiratory competent. To visualize mtDNA, cells were fixed either in 70% ethanol containing 10 ng/ml DAPI or with 3.7% formaldehyde for 1 h, followed by spheroplasting and incubation in PBS containing 1 μg/ml DAPI. Fixed, stained cells were washed into PBS and imaged using standard epi-fluorescence microscopy. To analyze mitochondrial morphology, aliquots of cells were also processed for indirect immunofluorescence and imaged using confocal microscopy (see below). The mutation in mgm101-2 was determined by double-stranded DNA sequence analysis. Yeast genomic DNA from strains JNY131 and W303 was prepared as described and used as template for polymerase chain reaction (PCR) with the following sets of primers corresponding to the MGM101 gene: 5′-CGGAATTCATGAAAAGCATTTTCAAGG-3′ and 5′-GCTCTAGACTATTTATAAGGATATTCAAC-3′, 5′-GGAATTCCTCCTTGGACAACACTTTCGT-3′ and 5′-CGG- GATCCCGCAACTTCTTTTGGATACCAG-3′ (BRL-Custom Primers Inc.). The PCR products from five independent reactions with a given set of primers were pooled, gel purified, and sequenced using the above listed primers by the Division of Biological Sciences Automated DNA Sequencing Facility at University of California, Davis. For the production of anti-Mgm101p and anti-Abf2p antibodies, maltose binding protein (MBP) fusions were created. MGM101 and ABF2 were cloned into pMAL-c2 ( New England Biolabs, Inc. ) using an in-frame 5′ EcoRI site and a 3′ XbaI site introduced using PCR. Fusion proteins were expressed in Escherichia coli (DH5 α) at 25°C and purified using amylose affinity chromatography following the methods outlined by New England Biolabs, Inc. Rabbit polyclonal antibodies were raised against fusion proteins by injecting 1 mg of purified protein subcutaneously every 2 wk. Anti-Mgm101p antibody was affinity purified as described . Highly enriched yeast mitochondria were prepared essentially as described with the following modifications. Cultures of either W303 or JNY131 were grown in YPG media to log phase. Before harvesting, yeast cell walls were removed using yeast lytic enzyme (ICN). Isolated crude mitochondrial pellets from lysed cells were resuspended in 50% w/v optiprep (Nycomed), 20 mM HEPES, pH 7.4, osmotically balanced with sorbitol to 600 milli-osmolal. This mixture was used as the bottom layer of optiprep step gradients formed in SW28 centrifuge tubes (Beckman); the layers atop it had densities of 1.1 g/ml and 1.16 g/ml, and were similarly osmotically balanced with sorbitol. Gradients were spun at 80,000 g for a minimum of 3 h in an SW28 rotor; mitochondria floated to the interface between the 1.1 and 1.16 g/ml. This interface was collected and diluted approximately fivefold with mitochondria isolation buffer (MIB: 0.6 M sorbitol, 20 mM Hepes, pH 7.4) and spun at 10,000 g for 10 min to pellet the mitochondria. This pellet was resuspended, centrifuged at 3,000 g for 5 min to pellet aggregated material and the supernate was centrifuged again at 10,000 g for 10 min and the pellet was resuspended in a minimal volume of MIB. Mitochondrial nucleoids were prepared using a modification of a technique described by Miyakawa et al. . Mitochondrial lysate was prepared by diluting purified mitochondria to a protein concentration of 2.5 mg/ml with nucleoid extraction buffer (NXB: 20 mM Hepes, pH 7.4, 100 mM sucrose, 20 mM KCl, 1% NP-40, 1 mM spermidine, 100 ng/ml DAPI, 1 mM PMSF, 0.5 mM DTT) followed by an incubation on ice for 1 h. This lysate was layered onto a 37.5%/60%/80% sucrose step gradient. In these gradients, the 37.5% layer contained NXB, and the 60% layer contained 20 mM Hepes, pH 7.4, 20 mM KCl, 1% Mega-8 ( Calbiochem ), 1 mM spermidine, 100 ng/ml DAPI, and 0.5 mM DTT. Gradients were spun at 100,000 g for 90 min; mitochondrial DNA-containing structures banded at the 37.5%/ 60% interface as assessed by fluorescence microscopy of DAPI stainable structures and enrichment of both DNA assayed using the picogreen reagent (Molecular Probes) and the nucleoid-associated protein Abf2p (see below). This material was collected and diluted twofold with NXB containing 2% NP-40 and rebanded over another 37.5%/60%/80% step sucrose gradient. To extract nucleoid-associated proteins from DNA, an equal volume of NXB containing 2 M KCl was added to the diluted nucleoid fraction. This mixture was incubated for 1–2 h on ice to produce a high salt extract enriched for nucleoid-associated proteins. This mixture was layered atop a 30% sucrose cushion containing NXB with 1 M KCl, and centrifuged at 100,000 g for 90 min. The top fraction, not including the top/30% interface, was collected. Protein in this fraction was precipitated by the addition of trichloroacetic acid to 15% wt/vol followed by centrifugation at 20,000 g for 30 min. The resulting pellet was rinsed twice with cold 80% acetone and then resuspended in freshly prepared 8 M urea, 100 mM ammonium bicarbonate, pH 8.0, at room temperature. After 12 h at 4°C, the mixture was centrifuged at 20,000 g for 30 min. The protein concentration in the supernatant from this spin, termed HSE, was determined using the Nano-orange reagent kit (Molecular Probes). DTT was added to HSE to 4 mM, and the mixture was incubated for 30 min at 37°C then diluted fourfold with 50 mM Tris-HCl, pH 8.0, 20 mM KCl, 1 mM CaCl 2 containing 1 μg of modified trypsin ( Boehringer ) for every 25 μg of protein. This mixture was incubated for 12 h at 37°C and then frozen in liquid nitrogen. Micro-column high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was used to identify the mixture of proteins present in the HSE fraction. The solution was loaded onto a 100 μm by 200 μm fused silica capillary (Polymetrics, Inc.) that was packed to a length of ∼15 cm with 10 μm POROS 10 R2 reverse phase material . The fritted end of the column was inserted into the needle of the Finnigan TSQ-700 electrospray ion source. The sample was directly loaded onto the micro column by helium pressurization of the sample in a stainless steel bomb . Solvent flow from a dual syringe pump (Applied Biosystems) was split 100:1 pre-column to deliver a final flow rate of 1 to 1.5 μm/min through the column. By using a mobile phase consisting of 0.5% acetic acid (solvent A) and 80:20 acetonitrile/water containing 0.5% acetic acid (solvent B) liquid chromatography was performed by ramping from 0% B to 60% B over 90 min. Electrospray ionization was carried out at a voltage of 4.6 kV and tandem mass spectra were acquired automatically during the entire gradient run . Each tandem mass spectrum was searched, using the SEQUEST program against the yeast orfs protein database obtained from the Saccharomyces Genome Database (Stanford University). Sequences for bovine trypsin and human keratin were included to facilitate identification of potential contaminants. Each high scoring peptide sequence, and the corresponding tandem mass spectrum, was manually inspected to insure the match was correct. Nucleoid-associated proteins were isolated from mitochondria and extracted from mitochondrial DNA according to the methods described above. Nucleoid-extracted proteins were dialyzed for 3 h at 4°C against a buffer containing 25 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM beta-mercaptoethanol. After dialysis, the extract was fractionated using native DNA cellulose chromatography as described . Cultures were grown either in YPD or YPG to early log phase. 5 OD 600 units were harvested by centrifugation and cells were washed once in distilled water. Cell pellets were resuspended in 75 μl SDS-PAGE sample buffer (2% SDS, 80 mM Tris-HCl, pH 6.8, 10% glycerol (vol/vol), 5% 2-mercaptoethanol, 1 mM phenylmethyl sulfonyl fluoride, 3 mM benzamidine). The mixture was boiled for 10 min at 100°C, and centrifuged for 15 min at 13,000 g . 5 μl of the supernatant was used for SDS-PAGE and immunoblot analysis. Proteins were analyzed using 17.5% SDS-PAGE, prepared according to . For Western blotting, anti-Mgm101p, anti-Abf2p, and anti-Por1p (Molecular Probes) antibodies were used at dilutions of 1:500, 1:1,000, and 1:500, respectively. Primary antibodies were incubated for a minimum of 1 h at 25°C, followed by incubation for a minimum of 30 min with secondary antibody at a dilution of 1:1000 (goat anti–rabbit HRP antibody and goat anti–mouse HRP antibody; Southern Biotechnology Associates, Inc.). Western blots were developed using ECL reagents ( Amersham ). To analyze mitochondrial morphology in strain JNY131, cells were shifted to nonpermissive temperature (37°C) and samples were taken at various time intervals and processed for indirect immunofluorescence. To localize Mgm101p, W303 was transformed with a 2μ plasmid containing MGM101 (kindly provided by G.D. Clark-Walker) and was grown in YPG at 30°C to log phase. At least 5 A 600 units of cells were fixed and processed for indirect immunofluorescence as described . Primary antibody incubation was for 1 h at room temperature with the following antibody dilutions into PBS/4% BSA: anti-Por1p at 1 μg/ml and affinity-purified anti-Mgm101p at 1:50. Fluorescein-conjugated anti-mouse secondary antibody or Texas red–conjugated anti-rabbit (Kappel) at a dilution of 1:100 in PBS/4% BSA was incubated with the cells at room temperature for 1 h, followed by three washes with PBS/4% BSA and one with PBS containing 1 μg/ml of DAPI. Mounting media (FITC-Guard; Testgog) was placed on fixed cells and coverslips were sealed with nail polish. All fluorescent samples were analyzed using a Leica confocal microscope and a 100× 1.4 NA objective. Figures were prepared using Adobe Photoshop and Adobe Illustrator. A GFP fusion to the NH 2 terminus of MGM101 was constructed by introducing 5′ and 3′ BamHI sites (in frame with the initiator ATG codon and after the termination codon) in MGM101 codon by PCR with Vent polymerase ( New England Biolabs ). A mitochondrially targeted (signal corresponding to bases 1–60 of COXIV) form of GFP downstream of the ADH promoter in plasmid pOK29 (kindly provided by Rob Jensen, Johns Hopkins, Baltimore, MD) was modified by introducing a BamHI site into the 3′ end of GFP(S65T) and eliminating the termination codon, resulting in pmitoNGFP. Modified MGM101 was ligated into pmitoNGFP, resulting in a gene encoding mitochondrially targeted NH 2 -terminal GFP fusion of Mgm101p expressed from the ADH promotor. Yeast cells expressing mito-GFPMgm101p were imaged vitally without fixation after incubation with 1 mg/ml DAPI for 30 min using a Leica confocal microscope and a 100× 1.4 NA objective. Thymidine kinase gene from Herpes virus was introduced into JNY131( mgm101-2 ) and W303 by crossing and sporulation. These strains, JNY131TK and W303TK, were cultured overnight in YPG at 25°C to 0.2/ml OD 600 units. Cells were washed twice and resuspended in 37°C YPD at same OD 600 unit/ml, and placed in 37°C. At fixed time points, a 5-ml aliquot (1 OD 600 unit) of each culture was removed, pelleted, and BrdU was incorporated into DNA by resuspension in 2 ml of YPD media containing 5 mg/ml sulfanilimide, 10 μg/ml amethopterin, and 0.5 mg/ml BrdU, and then incubated at 37°C for 30 min. BrdU incorporation into mtDNA was detected by indirect immunofluorescence as previously described . W303 and JNY131( mgm101-2 ) strains were grown at 25°C overnight in YPG to 0.2–0.3 OD 600 unit/ml. For UV-irradiation, both JNY131 ( mgm101-2 ) and W303 cells were washed into YPD and preincubated at 37°C for 2 h before irradiation. Cells were then plated onto YPD plates and exposed to UV radiation (Stratalinker; Stratagene) at doses ranging from 0–200 J/m 2 . For gamma-irradiation, overnight cultures of cells were washed twice, resuspended in 0.9% NaCl at 2 × 10 6 cells/ml, placed on ice and exposed to a Cs source for various times corresponding to 0–600 grays. For H 2 O 2 treatment, overnight cultures of cells were washed once in YPD, resuspended in YPD containing 0–20 mM H 2 O 2 and incubated at 25°C for 1 h. Cells were then harvested and washed twice in 20 mM potassium phosphate, pH 7.4. Immediately after exposure, cells were plated on YPD plates. After exposure to each mutagen, colonies were formed at a semi-permissive temperature of 34°C (4-d incubation) and respiratory competency was determined by colony color as described. In an effort to understand the mechanism underlying mitochondrial nucleoid organization and function, we have started to identify the proteins found in highly enriched (∼70-fold from total extracts) preparations of nucleoids using the technique of liquid chromatography coupled with tandem mass spectrometry . Using this technique, many individual proteins can be identified from a heterogeneous mixture of proteins, making it ideal for analyzing constituents of biological complexes . Tandem mass spectrometry analysis of trypsin-digested nucleoid-enriched fractions consistently identified several peptides contained in the protein encoded by the MGM101 gene . These biochemical data suggest that Mgm101p plays a direct role in mtDNA maintenance through an association with the mitochondrial nucleoid. We have also isolated a temperature sensitive allele of MGM101 , JNY131( mgm101-2 ), in a genetic screen for mutants that are unable to maintain mtDNA (see Materials and Methods; J. Wagner, E.D. Wong, and J. Nunnari, unpublished observations). To determine the specific mutation in mgm101-2 , we sequenced the mutated gene. Sequencing of the mgm101-2 allele revealed a single point mutation, resulting in a change of the conserved Asp131 residue to Asn . This residue is invariant in Mgm101p among all species and is found at the beginning of the highly conserved COOH-terminal region of the protein. Interestingly, this alteration is a relatively conservative change in Mgm101p's primary structure. The fact that it causes the catastrophic loss of mtDNA under nonpermissive conditions reinforces the importance of the COOH-terminal region in Mgm101p function. To gain more insight into the role of MGM101 in mtDNA maintenance, we have characterized the kinetics of mtDNA loss in JNY131( mgm101-2 ). Loss of respiratory competence was determined by plating JNY131( mgm101-2 ) cells onto YPD media at fixed time points after shifting to nonpermissive temperature. After exposure to nonpermissive conditions, colonies were formed at permissive temperature and respiratory competence was assessed by examining colony color. Respiratory competent cells containing an ade2 mutation accumulate a red pigment when adenine is limiting in the medium. Thus, on YPD plates, red colonies are classified as respiratory competent and white and sectored colonies are classified as respiratory deficient. This classification was confirmed by analyzing the growth of colonies replica plated on medium containing the nonfermentable carbon source, glycerol. Under permissive conditions, no difference in the generation of respiratory incompetent colonies was observed in JNY131 ( mgm101-2 ) and W303, indicating that mgm101-2 allele is functional under these conditions . After shifting to nonpermissive temperature, however, JNY131( mgm101-2 ) cells began to lose respiratory competence after 4 h (two doublings). With continued exposure to nonpermissive temperature, the number of nonrespiratory colonies increased until the entire population of JNY131( mgm101-2 ) cells were respiratory deficient at 12 h . In contrast, a substantially lower percentage (overall 10%) of W303 cells became respiratory deficient at nonpermissive conditions . The relatively rapid loss of respiratory competence in the JNY131( mgm101-2 ) strain under nonpermissive conditions is similar to that observed previously for mgm101-1 cells and reflects the importance of Mgm101p in mtDNA maintenance. To determine the nature of the nonrespiring phenotype in JNY131( mgm101-2 ), cells from nonrespiring colonies were crossed to wild-type cells lacking mtDNA. Diploids resulting from this cross were also respiratory deficient, indicating that loss of respiratory function was the result of a mutation (rho−) or complete loss (rho°) of mtDNA. To distinguish between rho− and rho° cells, mtDNA was examined directly in cells from nonrespiratory JNY131 ( mgm101-2 ) colonies by staining with the DNA-specific fluorescent probe, DAPI. Interestingly, after a nonpermissive exposure time that resulted in 100% nonrespiring colonies, only ∼50% of the nonrespiring colonies consisted of rho° cells . The remaining half of the colonies consisted of rho− cells that still possessed mitochondrial nucleoids. However, after a more prolonged exposure of JNY131( mgm101-2 ) cells to nonpermissive temperature (16 h), 100% of nonrespiring colonies consisted of cells that were rho° , consistent with the colony phenotype observed in the screen that identified JNY131( mgm101-2 ) (see Materials and Methods). These observations indicate that a transition occurs where nonfunctional mtDNA (rho−) is formed in JNY131( mgm101-2 ) cells before the complete loss of mtDNA (rho°). This phenotype is consistent with an early deficiency in JNY131( mgm101-2 ) cells that causes the degradation of mtDNA or results in a block in mtDNA replication. Because mtDNA loss is also observed in a sub-group of mutants that affect mitochondrial morphology, such as mgm1 , mdm10 , mdm12 , and fzo1 , mitochondrial structure in JNY131( mgm101-2 ) cells was assessed by indirect immunofluorescence using antibodies directed against Por1p, a mitochondrial outer membrane protein . Mitochondrial morphology was unchanged and was indistinguishable from the cortical, reticular structure of wild-type mitochondria in S . cerevisiae . Transmission of the mitochondrial organelle also was unaffected, even after exposure to nonpermissive temperature for 12 h . This indicates that loss of respiratory function and mtDNA in JNY131( mgm101-2 ) is not associated with mitochondrial morphology and transmission defects. Together with the biochemical identification of Mgm101p as a component of enriched nucleoid preparations, these data suggest that Mgm101p plays a more direct role in mtDNA maintenance. Both genetic and biochemical analyses suggest that Mgm- 101p is localized to the mitochondrial nucleoid. To determine the subcellular localization of Mgm101p, we raised polyclonal antibodies against a MBP-Mgm101p fusion protein. Western blot analysis of W303 whole cell extracts with anti-Mgm101p antibodies detected a band at 30 kD, the predicted molecular mass of Mgm101p . In cell extracts made from CS6-1D, which contains a recessive inactive truncated copy of MGM101 , a band at ∼20 kD was detected, corresponding to the predicted Mgm- 101p product formed by the chromosomal disruption in this strain . To determine whether Mgm101p was localized to the mitochondria, we fractionated whole cell extracts from W303( pep4 ) cells by differential centrifugation. Samples from each fraction were analyzed by SDS-PAGE and immunoblotted with anti-Por1p, a mitochondrial outer membrane protein, anti-Abf2p, a known mitochondrial nucleoid-associated protein, and anti-Mgm101p . After low speed centrifugation of total cell lysate , mitochondria were recovered in both the supernatant and pellet , as indicated by the detection of Por1p and Abf2p. The presence of mitochondria in the low speed pellet was probably due to incomplete cell lysis. A similar fractionation pattern was observed for Mgm101p. When the low speed supernatant was centrifuged at 9,000 g , Por1p and Abf2p again cofractionated and were recovered in the high speed pellet , consistent with the mitochondrial localization of these proteins. Mgm101p was also recovered and enriched in the pellet fraction, indicating that it is a mitochondrial protein . To determine whether Mgm101p was associated with mitochondrial nucleoids, the mitochondrial-enriched pellet was subjected to equilibrium density centrifugation and detergent extraction . Mitochondrial nucleoids were purified further by fractionation of the mitochondrial detergent extract on two successive sucrose density gradients . Samples from the second sucrose gradient were subjected to SDS-PAGE and immunoblotted with antibodies against Por1p, Abf2p, and Mgm101p. As indicated by the fractionation behavior of the nucleoid marker Abf2p, mitochondrial nucleoids migrated to the 37.5%/ 60% sucrose interface, consistent with previously published results . In contrast, Por1p did not migrate into the sucrose gradient, as indicated by its presence in the gradient's top fraction, confirming its nonnucleoid association and demonstrating the observed enrichment of nucleoid-associated proteins at the 37.5%/60% sucrose interface . Mgm101p cofractionated with Abf2p and was quantitatively recovered in the fraction representing the 37.5%/60% interface, suggesting that it is tightly associated with nucleoids . To confirm the biochemical fractionation data, we localized Mgm101p by tagging the protein at the NH 2 and the COOH termini with GFP. Fusion of mito-GFP to the NH 2 terminus of Mgm101p (mito-GFP-Mgm101p, see Materials and Methods) created a protein that when expressed from a constitutive promoter ( ADH ) in JNY131( mgm-101-2 ) cells could partially complement the temperature-sensitive growth phenotype on glycerol, indicating that it was functional. In addition, DAPI staining of nonrespiring JNY131( mgm101-2 ) cells expressing mito-GFP-Mgm101p revealed that they were rho−, in contrast to the rho° phenotype that results from mgm101-2 . This also indicates that mito-GFP-Mgm101p is able to partially rescue the defect in JNY131( mgm101-2 ) cells. Expression of a COOH-terminal fusion of GFP to Mgm101p failed to complement growth of JNY131( mgm101-2 ) on nonfermentable carbon sources. However, JNY131( mgm101-2 ) cells expressing Mgm101p-GFP were also 100% rho− under nonpermissive conditions. This indicates that Mgm101-GFP also retains some function. Interestingly, overexpression of either mito-GFP-Mgm101p or Mgm101p-GFP, but not wild-type Mgm101p, in W303 cells resulted in a significant increase in the formation of nonrespiratory cells (∼50-fold increase), suggesting that fusion of GFP at either terminus interferes with some process required for mtDNA maintenance. Visualization of either live wild-type or JNY131( mgm-101-2 ) cells expressing GFP-Mgm101p and Mgm101-GFP (data not shown) revealed that GFP was localized to punctate structures at the cortex of the cell that completely overlap with vitally DAPI-labeled mtDNA. Thus, in vivo, Mgm101p/GFP fusion proteins are localized to the mitochondrial nucleoids. Localization of Mgm101p in cells was also determined using indirect immunofluorescence with anti-Mgm101p antibodies. No staining above background was observed with anti-Mgm101p in wild-type cells (data not shown). However, in cells overexpressing Mgm101p, anti-Mgm-101p labeled punctate structures at the cortex of the cell . These punctate structures were also labeled with DAPI, indicating that they contained mtDNA. As stated above, overexpression of Mgm101p in wild-type cells had no observable effect on mtDNA maintenance or inheritance as compared with wild-type. It is important to note that because we could only detect Mgm101p when overexpressed in cells, we cannot rule out the possibility that, when present at wild-type levels, Mgm101p may localize to only a subset of nucleoids. However, these data are in agreement with data obtained from both the biochemical fractionation behavior of Mgm101p and the localization of the Mgm101p/GFP fusions and indicate that Mgm101p is localized specifically and uniquely to the mitochondrial nucleoid. The COOH-terminal region of Mgm101p is highly basic suggesting that Mgm101p has the ability to bind DNA. To determine if Mgm101p has DNA binding activity, nucleoids were isolated from W303 and associated proteins were extracted with high salt containing buffer, separated from mtDNA by sucrose gradient centrifugation and subjected to DNA-cellulose chromatography. Both nucleoid proteins Abf2p and Mgm101p were quantitatively recovered in this high salt nucleoid extract, indicating that their association with this structure is salt-sensitive. Given that this may reflect their association with mtDNA, the salt concentration in the extract was lowered to 200 mM before DNA-cellulose chromatography. Under these conditions, both Abf2p and Mgm101p were soluble and sedimented as monomers as determined by sucrose gradient sedimentation (data not shown). Abf2p bound to the DNA-cellulose column and could be eluted only at high salt concentrations (1 M), consistent with its known high affinity interaction with DNA . Mgm101p also bound and eluted quantitatively from the DNA cellulose at relatively high salt concentrations . This tight association of Mgm- 101p with DNA cellulose suggests that Mgm101p specifically interacts with DNA. Furthermore, both Mgm101p and Abf2p were significantly enriched, 60- and 100-fold, respectively, in DNA-cellulose column eluates. Taken together these data suggest that the localization of Mgm101p to the nucleoid is at least in part due to direct interaction with mtDNA. To gain more insight into MGM101 function, we examined Mgm101p under both permissive and nonpermissive conditions in JNY131( mgm101-2 ). Total extracts from JNY131( mgm101-2 ) and W303 cultures grown under both conditions were analyzed by SDS-PAGE and immunoblotting with anti-Mgm101p . In extracts from JNY131( mgm101-2 ) cultured under permissive conditions, a band was recognized specifically by anti-Mgm101p that migrated faster than Mgm101p . This faster migrating product is unlikely to be an in vitro isolation artifact because the identical migration behavior was observed when Mgm101-D131Np was examined from denaturing immunoprecipitation of Mgm101p from S35-Met pulse-labeled cells (data not shown). The faster migration of Mgm101p observed under permissive conditions is thus likely the result of an in vivo proteolytic modification that may contribute to the phenotype observed at nonpermissive temperature (see below). As a function of time at nonpermissive temperature, the amount of Mgm101-D131Np observed was reduced dramatically in JNY131( mgm101-2 ) cells . Specifically, reduction of Mgm101-D131Np content occurred after 4 h and Mgm101-D131Np was not detectable above background after 6 h of exposure to nonpermissive temperature. In contrast, the steady state level of Mgm- 101p was unaffected under these conditions (data not shown). This observed thermolability of Mgm101-D131Np slightly precedes and correlates with loss of respiratory function and mtDNA in JNY131( mgm101-2 ) observed at nonpermissive temperature and therefore is likely the primary cause of these phenotypes. We investigated the function of Mgm101p in mtDNA maintenance by characterizing changes in nucleoid morphology in JNY131( mgm101-2 ) during loss of mtDNA under nonpermissive conditions. Failure of nucleoid segregation, as observed in ΔMGT1 cells, causes the accumulation of fewer and brighter-stained nucleoid structures . Defects in mtDNA packaging, as observed in ΔABF2 cells, cause nucleoid staining to become more diffuse . We reasoned that such analysis would help distinguish between these possible explanations of loss of mtDNA in JNY131( mgm101-2 ). We visualized nucleoids using DAPI in fixed JNY-131( mgm101-2 ) and W303 cells collected at fixed time points after exposure to nonpermissive temperature. Up to 4 h of exposure to nonpermissive temperature, the majority of JNY131( mgm101-2 ) cells contained mitochondrial nucleoids that were indistinguishable from nucleoids observed in W303 cells . This is consistent with the fact that the majority of JNY131 ( mgm101-2 ) cells retained respiratory competence during this time . In JNY131( mgm101-2 ) cells exposed to greater than 4 h of nonpermissive temperature mitochondrial nucleoid morphology remained punctate, but the staining of the nucleoids with DAPI became less intense and fewer nucleoids were detected . No staining of nucleoids with DAPI was detected after 8 h of exposure time (Table II ). In contrast, no change in nucleoid intensity was observed in either JNY131( mgm101-2 ) cells cultured under permissive conditions or W303 cells exposed to nonpermissive temperature, indicating that the effect was specific to the temperature-sensitive mgm101-2 allele . This phenotype correlates with and is likely the result of the temperature-dependent decrease in Mgm101-D131Np levels observed in JNY131( mgm101-2 ) at nonpermissive temperature. The lack of significant change in nucleoid morphology in JNY131( mgm101-2 ) suggests that segregation and packaging of mtDNA are not significantly affected. Furthermore, nucleoid partitioning into daughter buds of JNY131( mgm101-2 ) cells was also unaffected . Although segregation, packaging and partitioning of mtDNA do not seem to be grossly affected in JNY131 ( mgm101-2 ) cells, we did observe a dramatic decrease in the intensity of DAPI nucleoid staining over time at nonpermissive temperature. Interestingly, DAPI staining of nucleoids became undetectable before complete mtDNA loss in JNY131( mgm101-2 ) as assessed by examining mtDNA in cells from nonrespiratory JNY131( mgm101-2 ) colonies , consistent with our interpretation that mgm101-2 may cause mtDNA loss as a result of a block in mtDNA replication or by causing mtDNA degradation. To determine whether loss of mtDNA in JNY131 ( mgm-101-2 ) was the result of a block in mtDNA replication, we monitored the incorporation of the thymidine analogue, 5-bromodeoxyuridine (BrdU) into mtDNA by indirect immunofluorescence as previously described . Towards this goal, we constructed JNY131 ( mgm101-2 ) and W303 strains that contained a chromosomal copy of an exogenous thymidine kinase (JNY131TK and W303TK, respectively). Mitochondrial DNA replication was monitored in JNY131TK and W303TK cells grown under both permissive and nonpermissive conditions by pulse labeling with BrdU. Punctate staining in both JNY131TK and W303TK cells was detected at the cell cortex by indirect immunofluorescence of labeled cells with anti-BrdU antibodies . This punctate staining was not detected in either W303 rho° cells or W303 that had not been labeled with BrdU, indicating that it was specifically the result of incorporation of BrdU into mtDNA . Under permissive conditions, no significant differences in BrdU incorporation into mtDNA between JNY131TK and W303TK were observed (data not shown). Exposure of cells to nonpermissive temperature up to 4 h did not cause a significant reduction in the number of JNY131TK cells containing BrdU-labeled mtDNA or the intensity of mtDNA labeling as compared with W303 cells . This nonpermissive exposure time correlates with the onset of loss of respiratory function and the decrease in nucleoid staining intensity observed with DAPI in JNY131( mgm101-2 ) . Thus, the lack of an observable change in BrdU incorporation into mtDNA in JNY131( mgm101-2 ) before these phenotypes suggests that a block in mtDNA replication in JNY131TK is not the cause of these defects. Even after exposure times where Mgm101-D131Np content was greatly reduced by Western analysis, we observed BrdU-labeled mtDNA in JNY131( mgm101-2 ) cells of equal intensities (measured by pixel intensity, n = 20) to BrdU-labeled mtDNA in W303 cells . However, the percentage of JNY131TK cells that contained any detectable BrdU-labeled mtDNA decreased with time (Table II ). Not surprisingly, this unlabeled population correlates with the JNY131( mgm101-2 ) respiratory deficient colonies that contained rho° cells , indicating that lack of BrdU-labeled mtDNA in this population of cells was a secondary consequence of mtDNA loss. Taken together, these data suggest that under nonpermissive conditions, loss of respiratory function and mtDNA in JNY131 is not caused by a block in ongoing mtDNA replication. Given that ongoing mtDNA replication is unaffected in JNY131( mgm101-2 ), we reasoned that the decrease in nucleoid staining by DAPI observed in JNY131( mgm101-2 ) might be the result of mtDNA degradation. Consistent with this interpretation is that, after exposure to nonpermissive temperature, JNY131( mgm101-2 ) cells recovered rho− mtDNA under permissive conditions before complete mtDNA loss . Observations made in other systems, such as E. coli , indicate that damaged DNA is susceptible to DNA degradation. Thus, one explanation for these phenotypes is that JNY131( mgm101-2 ) cells are deficient in the repair of damaged mtDNA. To determine if Mgm101p plays a role in mtDNA repair, we examined the sensitivity of mtDNA to DNA damage in JNY131( mgm101-2 ) and W303 strains by monitoring the percent formation of respiratory deficient colonies. To examine the role of Mgm101p in the repair of UV-damaged mtDNA, we compromised MGM101 function by pretreating JNY131( mgm101-2 ) cells for 2 h at nonpermissive temperature before irradiation. After UV irradiating JNY131( mgm101-2 ) and W303 cells at various doses, colonies were formed at the semi-permissive temperature of 34°C. Under these conditions, in the absence of irradiation, we observed only a slight increase (2%) in the percentage of nonrespiratory colonies formed by JNY131 ( mgm101-2 ) cells as compared with W303 cells . Upon UV-irradiation, however, a significant dose-dependent increase in the percentage of nonrespiratory colonies was observed in JNY131( mgm101-2 ) as compared with W303 , indicating a defect in UV-induced mtDNA repair in JNY131( mgm101-2 ) cells. In contrast to the sensitivity of JNY131( mgm101-2 ) cells to mtDNA damage, nuclear DNA damage, as assessed by DNA damage-induced cell death, is unaffected , indicating that Mgm101p functions specifically in the repair of mtDNA and not nuclear DNA. Both pretreatment at nonpermissive temperature and incubation at the semi-permissive temperature of 34°C were necessary to observe significant mgm101-2 -dependent sensitivity to UV-induced mtDNA damage, consistent with the fact that Mgm101-D131Np is functional under permissive conditions. To determine if the mtDNA repair defect in JNY131 ( mgm101-2 ) cells was specific to UV-induced types of DNA damage, such as the formation of pyrimidine dimers, we examined the sensitivity of JNY131( mgm101-2 ) to gamma ray–induced mtDNA damage. In contrast to UV-irradiation, no preincubation at nonpermissive temperature was required before gamma ray treatment to observe a significant mgm101-2 -dependent sensitivity to mtDNA damage. However, after exposure, both JNY131( mgm101-2 ) and W303 colonies were formed at the semi-permissive temperature of 34°C. Similar to what was observed in the case of UV treatment, gamma ray irradiation of JNY131 ( mgm101-2 ) caused an increase in mtDNA-specific damage as compared with that observed for W303 cells . However, two significant differences were observed between the two mutagenic treatments. First, W303 cells were more sensitive to UV than gamma ray–induced mtDNA damage . Given that we observed a significant induction of gamma ray–induced damage in JNY131( mgm101-2 ) cells, the resistance of wild-type cells to gamma ray–induced mtDNA damage suggests that they repair the types of damage caused by this treatment more efficiently than damage caused by UV treatment. Most importantly, JNY131( mgm101-2 ) cells were more sensitive to gamma ray–induced mtDNA damage than to UV-induced mtDNA damage . This observation suggests that Mgm101p functions primarily in the repair of mtDNA damage selectively caused by gamma ray irradiation. Ionizing radiation in the form of gamma rays results in a range of DNA damage, but the predominant form is oxidative damage to bases and oxidative damage to sugars, which results in single strand breaks in the DNA . The severe defect of JNY131( mgm101-2 ) cells in the repair of ionizing-induced mtDNA damage is consistent with a model in which MGM101 functions in the repair of oxidative damage. To test this model, we examined the sensitivity of JNY131( mgm101-2 ) cells to oxidative mtDNA damage caused by the oxidant H 2 O 2 . As shown in Fig. 8 , E and F, JNY131( mgm101-2 ) cells were hypersensitive to H 2 O 2 -induced mtDNA damage as compared with wild-type. As in the case of gamma ray–induced damage, no preincubation at nonpermissive temperature was required before treatment to observe the mgm101-2 -dependent hypersensitivity to mtDNA damage, but after treatment colonies were formed at the semi-permissive temperature of 34°C. The hypersensitivity to both gamma ray– and H 2 O 2 -induced mtDNA damage in JNY131( mgm101-2 ) cells indicates that MGM101 function is required selectively for the repair of oxidatively damaged mtDNA. Indeed, this repair defect in JNY131( mgm101-2 ) cells may be the cause of the catastrophic loss of mtDNA in JNY131( mgm101-2 ) cells at 37°C that have not been treated with DNA damaging agents. The mitochondrial nucleoid is a complex nucleoprotein structure that functions not only to package and store the genetic material, but also to organize the metabolic and segregational activities associated with its maintenance and inheritance. It is surprising that, with a few exceptions, both the constituents and the mechanisms involved in the assembly of this structure are relatively uncharacterized to date . The MGM101 gene was previously shown to be required for the maintenance of mtDNA, but its precise role remained unidentified . Our analysis of Mgm101p and the mgm101-2 mutant strain indicates that MGM101 's function is specific to the nucleoid structure and mtDNA. Loss of MGM101 function initially causes cells to rapidly lose respiratory function due to irreversible mutations in mtDNA (accumulation of rho− genomes) and ultimately causes the complete and catastrophic loss of mtDNA. This loss of mtDNA in mgm101-2 cells is not associated with any changes in the structure or organization of the mitochondrial organelle. This is in contrast to another group of mutants that cause mtDNA loss as a result of a primary defect in the maintenance of mitochondrial morphology . Using a number of independent approaches, we have also observed that Mgm101p is exclusively localized to nucleoids in cells. The nucleoid-specific localization of Mgm101p is probably due to its ability to bind tightly and specifically to DNA, suggesting that Mgm101p's function in mtDNA maintenance requires this DNA binding activity. Consistent with this is the fact that deletion of a small portion of the COOH terminus composed of 23% of positively charged residues results in loss of MGM101 function . What is the nature of Mgm101p's nucleoid-specific function? In vivo, nucleoid-specific functions required for mtDNA maintenance include the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. From our analyses of mgm101-2 cells, it is unlikely that Mgm101p is required for mtDNA packaging or segregation because in mgm101-2 cells, nucleoid morphology remained punctate and only a decrease in DAPI staining intensity of nucleoids under nonpermissive conditions was observed. It is important to note that our analysis of nucleoid morphology using DAPI might not have identified a subtle change in packaging or segregation of mtDNA. However, one would not expect a subtle change to cause the catastrophic loss of mtDNA observed in mgm101-2 cells. In addition, alterations in both the packaging and segregation of mtDNA have been previously observed using this technique. In cells lacking ABF2 , a nuclear gene that encodes a mtDNA packaging protein, nucleoids lose their punctate structure and instead possess a more diffuse morphology within the organelle . In cells lacking MGT1 , a gene that encodes a cruciform-cutting endonuclease, fewer nucleoids are observed and each contains a greater amount of mtDNA, as assessed by DAPI staining intensity . Furthermore, loss of MGM101 function did not cause a defect in the partitioning of nucleoids to daughter buds. To date, no mutants have been found that block the movement of mtDNA within the organelle. The observed decrease in nucleoid staining intensity with DAPI during loss of MGM101 function suggests instead that MGM101 is involved in another, more central process required for mtDNA maintenance, such as DNA metabolism. One key metabolic function required for mtDNA maintenance is replication. The decrease in nucleoid staining intensity with DAPI we observed in mgm101-2 cells is consistent with a decrease in nucleoid mtDNA content. We reasoned that this decrease could be the result of a block in mtDNA replication in mgm101-2 cells. Surprisingly, mtDNA replication, as monitored by BrdU incorporation in mgm101-2 cells did not appear to require MGM101 function. In contrast, we observed a rapid block (within 1 h at nonpermissive temperature) in mtDNA replication in a strain containing a temperature sensitive allele of MIP1 , the gene encoding mtDNA polymerase, that exhibited loss of respiratory function with kinetics similar to mgm101-2 cells (Meeusen, S., and J. Nunnari, unpublished data). This observation indicates that the sensitivity of our assay for mtDNA replication is sufficient to detect defects in mtDNA replication. Based on our analysis, however, we cannot rule out a potential role of Mgm101p in the initiation of mtDNA replication. For example, one might not expect to observe a decrease in BrdU incorporation if mtDNA were replicating via a rolling circle mechanism and initiation occurred before the loss of MGM101 function. Although unlikely, such a scenario might explain why we observe wild-type BrdU labeling intensity of mtDNA several generations after the complete loss of Mgm101-D131Np. Based on our data, the most likely model is that MGM101 functions in the repair of oxidatively damaged mtDNA. Most consistent with this interpretation, is that mtDNA in mgm101-2 cells is hypersensitive to damage caused by gamma ray irradiation and H 2 O 2 . These two agents induce DNA damage predominantly in the form of oxidative damage to bases and to sugars . In contrast, mgm101-2 cells were less sensitive to damage caused by UV treatment that induces oxidative DNA damage to a lesser extent . In addition, at the time of DNA damage by gamma ray and H 2 O 2 treatments, Mgm101-D131Np was functional in mgm101-2 cells, indicating that Mgm101p functions in the repair of damaged mtDNA and not in the protection of mtDNA from damage. In light of the probable role of MGM101 in mtDNA repair, the decrease in nucleoid DAPI staining intensity observed in mgm101-2 cells likely results from a transition where damaged mtDNA is degraded and ultimately lost, a phenomenon observed in other systems in response to unrepaired, damaged DNA . Our data also indicate that MGM101 functions exclusively in the repair of DNA contained in the mitochondrial organelle. It is interesting to note that mtDNA in wild-type cells is more resistant to treatments that produce oxidative damage, suggesting that yeast cells possess a greater ability to repair this type of mtDNA lesion . Thus, the repair pathway for oxidatively damaged mtDNA might be one of the major repair pathways in the mitochondrial organelle. Indeed, given the highly oxidative environment of the mitochondrial organelle it is possible that MGM101 functions exclusively in oxidative damage repair and that this function is essential for mtDNA maintenance. Alternatively, it is also possible that MGM101 function is not restricted to mtDNA repair and that its requirement for mtDNA maintenance reflects its role in a more fundamental DNA metabolic activity. What type of mtDNA repair pathway is Mgm101p involved in? As is the case for nuclear DNA, several different repair pathways function in the mitochondria. In yeast, there are at least two known repair pathways for mtDNA. One direct pathway is responsible for the repair of UV-induced pyrimidine dimers and is dependent on the PHR1 gene, which encodes a photolyase that functions in both the nucleus and the mitochondria . Given that MGM101 functions selectively in the repair of oxidatively damaged mtDNA, it is unlikely that it is involved in the direct PHR1-dependent photoreactivation repair pathway. Furthermore, the effects of UV-induced mtDNA damage in mgm101-2 cells were assessed in the dark and the PHR1 pathway is known to be dependent on light for activation . A mtDNA mismatch excision repair pathway has also been characterized in yeast and is dependent on the MSH1 gene, which encodes an ATPase that is homologous to eubacterial MutS mismatch repair protein . It is also unlikely that MGM101 is involved specifically in this pathway, because, while loss of MGM-101 function causes the complete loss of mtDNA, loss of MSH1 function results only in the accumulation of mutated copies of the mitochondrial genome, a substantially less severe phenotype . Thus, if MGM101 is involved in mismatch repair, it is likely to be required additionally for a process more central to mtDNA maintenance. Modification of nucleotide bases is the predominant form of DNA damage by oxidants . Thus, it is possible that Mgm101p functions in base excision and/or nucleotide excision repair pathways, the major pathways responsible for the repair of this type of DNA lesion. Mitochondrial base excision repair pathways have been characterized in other organisms, making it likely that such pathways will also exist in yeast mitochondria . However, to date, there is no evidence to suggest that mitochondria from any organism possess nucleotide excision repair pathways. Indeed, from a number of studies it has been suggested that mitochondria lack mechanisms to repair more bulky DNA lesions . Oxidative damage to sugar moieties in DNA also result in single strand breaks . If multiple single strand breaks occur in a localized area, the accurate repair of such a lesion would require a recombinational process . Thus, it is also possible that Mgm101p functions in such a repair pathway. Recombination-mediated repair pathways have not been described in detail in mitochondria . However, it is known that mtDNA is highly recombinogenic , suggesting that damaged mtDNA can be repaired in a manner dependent on this process. What molecular role does Mgm101p perform in mitochondrial DNA repair and maintenance? Clues to the molecular function of mitochondrial proteins can often be gained by searching for similarity among their origins in eubacterial proteins. However, it is interesting to note that structural homologues of mitochondrial proteins involved in DNA metabolism are also found in such evolutionarily diverse systems as eukaryotes and phage, suggesting that mtDNA metabolism has evolved significantly from its eubacterial ancestors. Mgm101p falls into a potentially novel class of evolutionarily distinct proteins. Although Mgm- 101p shows a high degree of conservation with other putative Mgm101p homologues in organisms as diverse as S . pombe , it exhibits no significant homology to any known proteins, nor does it possess any obvious enzymatic domains or motifs. Based on our findings that Mgm101p is essential for both mtDNA maintenance and repair and is able to bind directly to DNA, it seems probable that Mgm101p is directly involved in mtDNA metabolism. | Study | biomedical | en | 0.999996 |
10209026 | Cultures of sympathetic neurons from the superior cervical ganglia of newborn rat pups were prepared as follows. After dissection, the ganglia were treated with 0.25% collagenase and 0.25% trypsin for 15 min, and then triturated with a pasteur pipet into a single cell dispersion. The cells were then plated onto “special dishes” that were prepared by adhering a glass coverslip to the bottom of a 35-mm plastic petri dish into which had been drilled a 1-cm-diam hole. For the preparation of these dishes, we used glass coverslips that had been photoetched with a pattern of demarcated boxes that assist in the relocation of individual cells (Bellco Glass, Inc.). Before plating the cells, the glass-bottomed well of the special dish was treated for 3 h with 1 mg/ml polylysine, rinsed extensively, and then treated with 10 μg/ml laminin ( Sigma Chemical Co. ) for 4 h. Cells were plated in medium consisting of Leibovitz' L-15 ( Sigma Chemical Co. ) supplemented with 0.6% glucose, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum (Hyclone), and 100 ng/ml nerve growth factor (Upstate Biotechnology Inc.). Most of the experiments and immunolocalization studies were performed on freshly plated neurons. Some of the immunofluorescence analyses were performed on older cultures, and, for these studies, we used a medium better suited to long-term culture . Western blots were used to document the presence of the katanin subunits in sympathetic neurons, and to test the specificities of the katanin polyclonal antibodies. Analyses were performed on superior cervical ganglia obtained from rat fetuses or pups at various ages. Electrophoresis and immunoblotting were performed as previously described . Immunofluorescence microscopy was used to determine the distribution of both katanin subunits in cultured neurons at various stages of development. For these analyses, neurons were either fixed directly or preextracted in a microtubule-stabilizing buffer before fixation. Fixation was either in −20°C methanol or 4% formaldehyde. Cultures were then treated for 30 min in a blocking solution containing 2% normal goat serum and 1% BSA in PBS, and exposed overnight at 4°C to one of the two primary antibodies (used at 1:100) diluted in blocking solution. The primary antibodies were the affinity-purified rabbit polyclonal antibodies specific for either the 60- or 80-kD subunit of katanin . The following morning, the cultures were rinsed three times in PBS, treated again for 30 min in blocking solution, exposed for 1 h at 37°C to an appropriate Cy-3–conjugated second antibody (purchased from Jackson ImmunoResearch Laboratories, Inc.) diluted in blocking solution, rinsed four times for 5 min each in PBS, and mounted in a medium that reduces photobleaching. To optimize the possibility of visualizing any potential association of katanin with specific cytoplasmic structures, optical sections were acquired using the 410 Laser Scanning Confocal Microscope ( Carl Zeiss, Inc. ). The optical sections were 0.5 μm in width, and all sections comprising an individual cell were examined. Images were depicted in “glow-scale pseudocolor,” which displays the highest fluorescence intensity in white, the lowest in red, and intermediate intensities in shades of yellow and orange . Immunoelectron microscopy was used to obtain a higher resolution perspective on the distribution of katanin in cultured neurons, and also to determine whether a portion of the katanin is localized to the region of the centrosome. For these analyses, we used the same procedure that we previously used to localize gamma tubulin in cultured neurons . The antibody against the 60-kD subunit was used for these analyses at a concentration of 1:50. The negatives obtained from the electron microscope were scanned with a flat-bed scanner to obtain a digitized image, after which each gold particle was manually highlighted with the pencil tool in Adobe Photoshop to enhance the contrast of the particles. For most centrosomes observed, we obtained a rough estimate of the number of microtubules attached to it and the number of gold particles associated with it. Estimates were obtained by counting and adding together the microtubules and gold particles observed on available sections. For immunofluorescence visualization of microtubules, we used our previously described procedure . Cultures were rinsed briefly in the microtubule-stabilizing buffer termed PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl 2 , pH 6.9), and then extracted for 3 min with 0.5% Triton X-100 in PHEM containing 10 μM taxol (provided as a gift from the National Cancer Institute). This treatment removes unassembled tubulin while preserving microtubules . The cultures were then fixed by the addition of an equal volume of PHEM containing 8% paraformaldehyde and 0.3% glutaraldehyde. After 10 min fixation, the cultures were rinsed twice in PBS, treated three times for 5-min each in PBS containing 10 mg/ml sodium borohydride, and rinsed again in PBS. Cultures were then treated for 30 min in a blocking solution containing 2% normal goat serum and 1% BSA in PBS, and exposed overnight at 4°C to the primary antibody diluted in blocking solution. The following morning, the cultures were rinsed three times in PBS, treated again for 30 min in blocking solution, exposed for 1 h at 37°C to an appropriate Cy-3–conjugated second antibody (see above) diluted in blocking solution, rinsed four times for 5 min each in PBS, and mounted in a medium that reduces photobleaching. The primary antibody was the mouse monoclonal antibody against β tubulin purchased from Amersham Corp. and used at 1:500. To investigate whether microtubules were attached to the centrosome in control neurons and neurons that had been injected with the function-blocking katanin antibody (see below), we used standard transmission electron microscopy. Cultures were first extracted by the same procedure used in the immunofluorescence analyses because we have found that extraction significantly enhances the clarity of microtubules in the electron micrographs. For these analyses, neurons were extracted, fixed, prepared, and visualized as described in an earlier report from our laboratory . In one set of experiments, a volume of roughly 4 pl of the function-blocking antibody against the 60-kD subunit of katanin was microinjected into cultured sympathetic neurons at a concentration of 6 mg/ml. Cells were microinjected roughly 45 min after plating, before the outgrowth of any neuronal processes. (Some cells already had short processes by this time, but these cells were not selected for microinjection.) At this point, the cultures were returned to the incubator for 6 h, and then prepared for immunofluorescence visualization of microtubules. In another set of experiments, we took advantage of a pharmacological regime that reveals the outward transport of microtubules from the centrosome . In this regime, nocodazole ( Aldrich Chemical Co. ) is introduced into the cultures 30 min after plating at a final concentration of 10 μg/ml, and the cultures are returned to the incubator. After 6 h, the cultures are rinsed twice with warm drug-free medium, placed in a third rinse of warm drug-free medium, and then returned to the incubator. After 3 min of recovery, vinblastine sulfate ( Sigma Chemical Co. ) is added to a final concentration of 50 nM, and the cultures are once again returned to the incubator for 30 or 60 min. Cultures are then prepared for immunofluorescence visualization of microtubule distribution. In the present study, we wished to determine the effects on microtubule distribution of inhibiting katanin function. To accomplish this, we microinjected the function-blocking katanin antibody either immediately after adding the nocodazole or roughly 1 h before removing the nocodazole. In some experiments, we microinjected a function-blocking antibody to a protein called centrin, which is also present at the centrosomes of many cell types and has been proposed as a candidate for releasing microtubules from the centrosome . This antibody was prepared and characterized as previously described , and was microinjected in the same manner as the katanin antibody. The antibody was provided as a kind gift from Dr. Jeffrey Salisbury of the Mayo Clinic (Rochester, MN). To obtain information on the relative lengths of the microtubules within the cell bodies of control and antibody-injected neurons, we quantified the total amount of microtubule polymer and the number of free ends of microtubules. To quantify the total amount of microtubule polymer, we extracted and fixed cultures 4–5 h after a portion of the neurons in the culture had been microinjected with the katanin antibody. Total microtubule levels in the cell body were quantified using MetaMorph software ( Universal Imaging Corp. ), and expressed in arbitrary fluorescence units (AFUs). 1 For illustrative purposes, fluorescence images were converted to a standard pseudocolor scale in which red represents the highest and purple the lowest intensity. To quantify the number of free ends of microtubules, we microinjected roughly 4 pl of rhodamine-labeled tubulin at a concentration of 4 mg/ml into the cell bodies of uninjected neurons and neurons that had been injected with the katanin antibody 4–5 h before the injection of the tubulin. We then waited 1 min to permit the incorporation of fluorescent tubulin onto the ends of the microtubules, after which the cells were extracted in our standard microtubule-stabilizing buffer (see above), and rapidly imaged with a cooled CCD (see below). For these analyses, images were acquired using a cooled CCD to visualize the entire cell body and to optimize resolution. The number of microtubule ends was obtained by counting individual fluorescent points from images displayed on a high-resolution computer monitor. Western blot analyses were performed to determine whether katanin is expressed in vertebrate neurons and to confirm the specificity of the antibodies. Because katanin is a relatively low-abundance protein, analyses were performed directly on samples of superior cervical ganglia, rather than on cultures generated from the ganglia. The blots reveal the presence of both 60- and 80-kD subunits of katanin within the developing ganglia . A single band for each subunit was detected in samples from superior cervical ganglia obtained from embryonic (E18), newborn, and 4-d rat pups. The bands were entirely similar to those obtained from cultured HeLa cells, which were used as a positive control. No other bands appeared within these samples, nor were any bands detected in control blots incubated without the primary antibody. Given that neurons are the principle cells that compose these ganglia (especially at E18), these results strongly suggest that katanin is expressed in neuronal cells, and demonstrate that the antibodies do not cross-react with other proteins expressed by neurons or nonneuronal cells within the ganglia. Immunofluorescence and immunelectron microscopy on cultures derived from the ganglia confirm that the neurons express both the 60- and 80-kD katanin subunits. Immunofluorescence analyses were performed on freshly plated neurons , and on neurons that had been permitted to develop in culture for 1 (not shown), 3 , or 7 (not shown) d. Similar results were obtained whether we used the antibody to the smaller or larger katanin subunit. All images presented in the figures were obtained using the antibody to the smaller subunit. Optical sections were obtained with the confocal microscope to assist in determining whether any particular regions within the cell body stained more intensely for katanin, and also to permit a better comparison of fluorescence intensities within the cell body and the processes. Images were depicted in glow-scale pseudocolor to further assist in determining whether there was a focal enrichment of katanin in the cell body that might correspond to the centrosome. In all of these cases, the immunofluorescence images showed a widespread distribution of both katanin subunits throughout the neuron. Katanin immunoreactivity was detected in the cell body and in all regions of developing axons and dendrites at all stages of development. The appearance of the staining was generally diffuse, suggesting that neurons might contain a soluble pool of katanin as well as a pool bound to structures such as the centrosome. However, the immunofluorescence images were entirely similar in neurons that had been extracted before fixation in our standard microtubule-stabilizing buffer supplemented with various concentrations of detergents such as Triton X-100 and saponin (data not shown). These results suggest either that virtually all of the katanin is bound to detergent-insoluble structures, or that soluble katanin precipitates under these extraction conditions. No enrichment at any discrete site within the cell body was observed, indicating that katanin is not specifically concentrated at the centrosome. The images suggest that katanin is present at higher concentrations within the cell body compared with axons or dendrites. However, this difference may not be as dramatic as it appears in the micrographs because even the thinnest optical section of the cell body is thicker than the width of a neuronal process. Entirely similar results were obtained in cultures fixed with aldehydes rather than cold methanol. No detectable staining was observed in cultures prepared with the secondary antibody but not the primary antibody. It has been our experience that immunofluorescence microscopy is not well suited to visualizing the centrosome in the near-spherical cell body of the neuron, even with the use of optical sectioning. For example, gamma tubulin is localized to the centrosome in neurons, but we have found that with immunofluorescence staining for gamma tubulin the centrosome appears as a small speckle that is difficult to distinguish from background speckles. Therefore, we have come to rely on immunoelectron microscopy as the best approach to determine with confidence whether a protein is associated with the centrosome . To investigate whether a portion of the katanin in the neuron is associated with the centrosome (even though the protein does not appear to be specifically concentrated at the centrosome), we analyzed the neurons with immunoelectron microscopy. In all neurons examined ( n = 18), katanin immunoreactivity was detected within or around the pericentriolar material . In addition, katanin immunoreactivity was sometimes associated with the clusters of amorphous cytoplasmic material that persist extraction . Occasionally, immunoreactivity was found at a discrete site along the length of a microtubule . Immunoreactivity was also found in axons and dendrites (not shown). Only an extremely rare gold particle was ever detected in cultures prepared in a similar fashion in the absence of the primary antibody, indicating the specificity of the results obtained with the primary antibody. Taken together, these immunological analyses demonstrate that katanin is expressed in neurons, that it has a widespread distribution throughout the cytoplasm, that it can associate with microtubules, and that a portion of the katanin is associated with the centrosome. It has been suggested that the presence of katanin at the centrosome might depend on having an array of attached microtubules . On this basis, it seems reasonable that a portion of the katanin might move to the centrosome shortly after a burst of microtubule nucleation, and then dissipate from the centrosome after the microtubules are released . To investigate this possibility, we compared the levels of katanin present at centrosomes with few or no attached microtubules with the levels present at centrosomes with higher numbers of attached microtubules. The vast majority of the centrosomes that we observed had few or no microtubules attached to them, and these centrosomes displayed roughly 30–50 gold particles (see Materials and Methods). A small fraction of the centrosomes we examined (2 of 18) showed roughly 15–20 attached microtubules, but these centrosomes did not display any higher immunoreactivity for katanin than centrosomes with few or no attached microtubules . Thus, the present observations do not support the idea that the levels of katanin at the neuronal centrosome vary as a result of the number of microtubules attached to it. In a first set of experimental analyses, freshly plated neurons were microinjected with the polyclonal antibody against the 60-kD subunit of katanin. This antibody has been shown by in vitro analyses to interfere with the microtubule-severing activity of katanin . Typical uninjected neurons grew an average of 633 ± 229 μm of total axon length during the first 6 h of plating. 4 of 50 neurons observed showed little or no outgrowth, presumably due to damage during plating. Neurons injected with katanin antibody that had been boiled before injection showed an entirely similar pattern of axon outgrowth, with 23 of 27 neurons each displaying levels of axon outgrowth comparable to controls (659 ± 108 μm total axon length). In sharp contrast, neurons injected with the viable katanin antibody were severely stunted with regard to axon outgrowth. A total of 45 such neurons were examined. The average total length of axons extended by these cells was 73.1 ± 73 μm. Fig. 3 A shows tracings of typical control neurons, while B shows tracings of typical katanin-antibody–injected neurons. Notably, neurons injected with a function-blocking antibody to centrin (another protein that has been suggested as a mediator of microtubule-release from the centrosome) showed no dramatic inhibition of process outgrowth. 20 cells injected with the centrin antibody grew an average of 569 ± 79 μm of total axon length. These data are summarized in Fig. 4 . Fig. 3 C shows an immunofluorescence micrograph of the microtubule array within a typical control (uninjected) neuron. There is a widespread and relatively even distribution of microtubules throughout the cell body. Neurons injected with the boiled katanin antibody or the centrin antibody were indistinguishable from uninjected neurons (not shown). Fig. 3 D shows a neuron that grew no processes after injection with the viable katanin antibody. Microtubules appear throughout the cell body, but, as with the uninjected cells, it is impossible to discern whether or not the microtubules are attached to the centrosome (even with optical sectioning at the confocal microscope; data not shown). Fig. 3 E shows a neuron that has extended short processes after injection of the viable katanin antibody. The microtubules have reorganized such that it is clear that several microtubules are attached to a “point source” in the cell body, presumably the centrosome. In addition, these microtubules are unusually long, extending to the periphery of the cell body. Fig. 3 F shows a neuron that has grown somewhat longer processes after injection of the viable katanin antibody. In this cell, it is not possible to discern the attachment of microtubules to a point source, but it is clear that many of the microtubules are clustered together near the center of the cell body. In addition, some of the individual microtubules appear to be unusually long. As noted above, immunofluorescence labeling is not well suited to identifying the centrosome of neuronal cells with confidence. The centrosome is very small and usually difficult to distinguish from background speckles, and there is usually insufficient resolution to visualize whether or not microtubules are actually attached to it. Therefore, electron microscopy was used to better evaluate the relationship between the microtubules and the centrosome within these control and experimental neurons. As previously reported , few or no microtubules are attached to the centrosome in control neurons . We previously speculated that this was due to the rapid release of microtubules after their nucleation . Indeed, neurons injected with the viable katanin antibody showed numerous microtubules attached to the centrosome. Fig. 5 , C and D show one such neuron (4–5 h after injection of the antibody). There are 18 microtubules attached to the centrosome in the thin section showed in the figure, and similar numbers in each of the other thin sections. We estimate that roughly 50–75 microtubules accumulated at this centrosome over the 4–5-h period of time. Similar results were obtained in all six such cells examined by electron microscopy. In the most dramatic case, 75–100 microtubules accumulated at the centrosome over the 4–5-h period of time (not shown). The fact that process outgrowth was compromised in the presence of the katanin antibody is consistent with the view that the released microtubules are essential for process outgrowth. However, the immediacy of the response was somewhat surprising given that the neuronal cell body contains a large number of microtubules that are not attached to the centrosome, presumably because they have already been released. Thus there is a “storage supply” of microtubules that can be transported into developing processes, and must be depleted from the cell body before axon outgrowth would be notably compromised by inhibiting the manufacture of new microtubules at the centrosome . For this reason, the immediacy of the inhibition of axon outgrowth by the katanin antibody suggests that katanin serves another important function in the neuron besides releasing microtubules from the centrosome. One possibility is that released microtubules must be continuously severed by katanin to keep them sufficiently short that they can effectively funnel into developing processes. If this is correct, we would expect the microtubules to be substantially longer in the antibody-injected cells compared with the control cells. On the basis of the immunofluorescence images shown in Fig. 3 , it was already our impression that some of the microtubules in the antibody-injected cells were unusually long, but it was impossible to draw a conclusion on this basis alone given that we could not discern individual microtubules in the control neurons. Unfortunately, the near-spherical geometry of the neuronal cell body precluded our ability to directly measure microtubule lengths using the same kind of serial reconstruction electron microscopy that has previously proved effective in such analyses on developing axons . For this reason, we used an indirect method to compare microtubule lengths. Specifically, we reasoned that we could indirectly compare microtubule lengths by determining the total microtubule mass in the cell body and the number of free microtubule ends. To quantify the total microtubule mass, we prepared cultures for immunofluorescence visualization of microtubules 4–5 h after a portion of the neurons had been injected with the katanin antibody. We found that the cell bodies of antibody-injected cells contained 647 ± 54 AFUs (4–5 h after antibody injection), while the cell bodies of uninjected cells contained 438 ± 55 AFUs ( n = 32 in each case). These data indicate that there is roughly a 48% increase in the levels of microtubule polymer in the cell bodies of the antibody-injected neurons compared with control neurons. Such an increase is consistent with an inhibition of microtubule severing, given that microtubule severing probably results in the depolymerization of a portion of the polymer . Fig. 6 , A and B, shows immunofluorescence images of a control and injected neuron, respectively, in a standard pseudocolor range to illustrate the fact that the cell bodies of antibody-injected neurons contained more polymer than control neurons. To quantify the number of microtubule ends, we microinjected rhodamine-labeled tubulin into the cell bodies of uninjected neurons and neurons that had been injected with the katanin antibody 4–5 h before the injection of the tubulin. We then waited 1 min to permit the incorporation of fluorescent tubulin onto the free ends of the microtubules, after which the cells were extracted in a microtubule-stabilizing buffer and rapidly imaged with a cooled CCD (see Materials and Methods). 30 s proved to be too short of a time to reveal much detectable incorporation of fluorescent tubulin, while 2 and 3 min proved to be too long, resulting in incorporation along substantial lengths of the microtubules. The 1-min time point was chosen because it produced a “speckled” appearance of free microtubule ends that could be quantified by counting the speckles on the computer monitor. In 10 control cells, we observed 448.3 ± 97 free microtubule ends , while in 10 antibody-injected cells we observed 96.1 ± 42 free microtubule ends . These results indicate that the average microtubule length was increased by at least four to five times (and probably even more given the greater microtubule mass) in the cell bodies of neurons in which katanin function had been inhibited for 4–5 h. The data for these studies are summarized in Fig. 7 . Another set of experimental analyses was aimed at more directly visualizing the effects of inhibiting microtubule release from the centrosome with the katanin antibody. These analyses were based on a pharmacologic assay that we used in earlier studies in which we documented that cytoplasmic dynein is the motor protein that conveys microtubules from the centrosome into the axon . For this assay, freshly plated neurons are treated with 10 μg/ml nocodazole for 6 h to depolymerize most of the microtubule polymer, after which the drug is removed for 3–5 min to permit a burst of synchronized microtubule assembly from the centrosome. Then, to prevent further microtubule assembly from occurring, 50 nM vinblastine is added. Under these conditions, showed schematically in Fig. 8 A, the microtubules are rapidly released from the centrosome and conveyed to the cell periphery in a dynein-dependent manner. Thus, this regime is useful for studying the outward transport of microtubules from the centrosome. Here, we injected the katanin antibody shortly after the addition of nocodazole. Fig. 8 B shows an uninjected cell; microtubules are concentrated around the periphery of the cell body 1 h after the addition of vinblastine. The same result was obtained with cells injected with the boiled katanin antibody (not shown). Fig. 8 , C–E, shows three examples of cells that were injected with the viable antibody. 1 h after addition of the vinblastine, in all cases the microtubules remained clustered within a discrete region of the cell body, and did not distribute around its periphery. In some cases, a clear point of attachment could not be clearly discerned , but in most cases the attachment of the microtubules to the centrosome could be discerned even at the immunofluorescence level . Interestingly, under these experimental conditions, the entire centrosome/microtubule complex often relocated from cell center to cell periphery. This relocation, which occurred in 16 of 29 total neurons examined, is presumably the result of the same motor-driven forces that normally convey individual microtubules to the cell periphery. Entirely similar results were obtained in a small number of experiments in which the katanin antibody was introduced during the first hour in nocodazole rather than the last hour (data not shown). The mechanisms that establish the neuronal microtubule arrays have been intensely studied for many years, but remain controversial. Studies from our laboratory support a model in which microtubules destined for axons and dendrites arise within the cell body of the neuron. In this model, the microtubules are nucleated by the centrosome and are oriented relative to their polarity by the motor proteins that convey them into axons and dendrites. In previous studies, we documented that gamma tubulin within the centrosome is essential for nucleating these microtubules , and that cytoplasmic dynein and CHO1/MKLP1 are the motor proteins that transport microtubules with their plus- or minus-ends leading, respectively, into neuronal processes . In the present study, we sought to test whether katanin is the protein responsible for severing (and thereby releasing) microtubules from the neuronal centrosome. We originally proposed that katanin might play this role because it is an ATPase with potent microtubule-severing properties that has been localized to the centrosome in other cell types . In the present study, we have documented that katanin is expressed in developing neurons, and that it is present throughout all regions of the neuronal cytoplasm, including the centrosome. In addition, we have performed functional studies in which we microinjected into cultured neurons an antibody that inhibits the capacity of katanin to sever microtubules. These studies demonstrated that katanin is essential for releasing microtubules from the neuronal centrosome, and for regulating the lengths of microtubules throughout the cell body of the neuron. Injection of the antibody also inhibited process outgrowth, suggesting that katanin-mediated events are also crucial for the normal deployment of microtubules into developing neuronal processes. Our initial expectation was that katanin would be concentrated more highly at the centrosome than in other locations in the neuron, or that the katanin that was not localized at the centrosome would be unbounded and lost during extraction. However, neither of these expectations proved to be the case. While katanin was always found at the centrosome, the amounts of the protein were not appreciably higher than those found elsewhere in the cell body. The fact that the noncentrosomal katanin is not detergent-extractable might suggest that it is associated with insoluble cytoplasmic structures. However, the identity of these structures is unclear from our micrographs, and there is always the possibility that the detergent extraction itself could have altered the normal association of katanin with specific cytoplasmic structures. In our preparations, katanin was typically found in association with amorphous cytoplasmic material, and was sometimes (but not often) observed on microtubules. It is possible that the katanin that was observed on microtubules might have been in the process of severing those microtubules at the time of fixation. However, the widespread distribution of the protein (even into the microtubule-free regions of the cell) indicates that much of the katanin is not microtubule associated. Previous studies suggested that the presence of microtubules at the centrosome might be required to target katanin to the pericentriolar material . However, in neurons, we found no relationship between the number of microtubules attached to the centrosome and the levels of katanin associated with it. Instead, the neuronal centrosome contains a fairly consistent amount of katanin at all times, which suggests that additional regulatory factors (rather than the absolute levels of katanin) probably determine the rate and degree of microtubule severing that occur at the centrosome at any moment in time. The fact that katanin is present throughout the neuron was our first indication that this protein may serve other functions in addition to releasing microtubules from the centrosome. It is well documented that the lengths of individual microtubules are tightly regulated within different regions of the neuron , but it has been assumed that microtubule length is principally regulated by dynamic assembly and disassembly events. One exception to this assumption was a study in which we showed that microtubule fragmentation occurs during collateral branch formation to transform a smaller number of long microtubules into a larger number of short microtubules . We hypothesized that a microtubule-severing protein such as katanin might be responsible for this fragmentation. Our present results are consistent with this hypothesis, but also show that microtubule severing by katanin might be a critical factor in regulating microtubule lengths throughout all regions of the neuron. Indeed, microinjection of the katanin antibody caused an inhibition of axon outgrowth that was far too rapid and dramatic to be attributed entirely to cessation of microtubule release from the centrosome. Neuronal cell bodies contain a “storage supply” of already-released microtubules that can support process outgrowth for several hours even in the absence of the production of new microtubules from the centrosome . On the basis of these results, it seems reasonable that microtubule severing by katanin might be necessary to keep microtubules in the cell body sufficiently short that they can funnel effectively into axons and dendrites. Indeed, analyses on the number of free microtubule ends confirmed that, after 4–5 h in the katanin antibody, the average length of the microtubules in the neuronal cell body was at least four to five times greater than in control neurons. On the basis of these very different microtubule arrays, we propose the following model for the effects of katanin inhibition on process outgrowth. In the control cell, microtubules are nucleated from the centrosome and are rapidly released by katanin after having obtained a length of no more than a few microns. At this point, motor proteins transport the microtubules outward toward the cell periphery. The microtubules undergo dynamic assembly and disassembly events, but they also undergo severing events, and these severing events are essential for ensuring that the microtubules remain relatively short. The transport machinery is able to effectively direct the ends of these short microtubules into the hillock regions of developing processes. By contrast, the experimental cells with inactive katanin do not release microtubules from the centrosome and hence do not have a continuous supply of new microtubules to support process outgrowth. In addition, unlike in control cells, the number of individual microtubules cannot be increased by severing the microtubules that had already been released. Moreover, without functional katanin, the already-released microtubules in the cell body rapidly obtain lengths that are simply too great to be effectively directed into developing processes by motor proteins. For all of these reasons, there is a rapid and dramatic inhibition of process outgrowth when katanin is experimentally inactivated. It should be acknowledged that there are other models for process outgrowth that do not invoke the transport of microtubules from the cell body. Such models might also be consistent with a crucial role for the severing of microtubules by katanin. For example, if one believes a model in which microtubules cannot be transported into processes from the cell body, microtubule severing by katanin could be invoked as the sole means to generate new microtubules for axons and dendrites (by severing preexisting microtubules within the processes). However, in our opinion, there is now unequivocal evidence indicating that microtubules are indeed transported into developing processes from the cell body . Thus we feel that the most reasonable interpretation of the present observations is that katanin releases microtubules from the centrosome, and then regulates their lengths after release so that they can be effectively transported into developing processes. However, given its widespread distribution, it seems reasonable to suggest that microtubule severing by katanin may also help to regulate microtubule number and length within the processes themselves, especially during the formation of branches. As previously noted, katanin is expressed in a wide variety of cells and is presumably also responsible for microtubule severing at the centrosome in these cells. However, most interphase cells do not release microtubules from the centrosome as rapidly or as completely as neurons. Even interphase epithelial cells, which establish microtubule arrays at sites distal to the centrosome, show significantly less active microtubule release than we have observed in neurons . It has been proposed that katanin may be particularly active during mitosis, during which time the minus ends of many of the microtubules that comprise the spindle must be released from the duplicated centrosomes for there to be a flux of tubulin subunit through these microtubules. At present, the factors that regulate katanin activity during the cell cycle are unknown, but our results suggest that the activity of katanin in terminally postmitotic neurons may be more similar to that in mitosis than during interphase. This is not a surprising conclusion, however, given that several recent studies from our laboratory indicate that many of the mechanisms that establish the microtubule arrays of the neuron are variations on analogous mechanisms that organize microtubules during mitosis . The widespread distribution of katanin at all stages of neuronal development suggests that the protein is not continuously active. As mentioned earlier, katanin is probably under the regulation of factors that locally activate or deactivate its microtubule-severing properties. It may be that microtubule release from the centrosome occurs in “pulses” determined by such activation and deactivation. If this is true, the frequency and/or timing of these pulses might be an important factor in targeting the appropriate number of microtubules to their appropriate locations during neuronal development. Whatever the mechanisms are that regulate katanin activity in the neuron, it seems reasonable that they would be similar to the factors that regulate the activity of the protein during the stages of the cell cycle of dividing cell types. | Study | biomedical | en | 0.999996 |
10209027 | An At-Rac1 cDNA probe was used to screen an A . thaliana whole-plant Uni-ZAP XR cDNA library (Stratagene). From phages containing hybridizing sequences, pBluescriptSK − phagemids were excised according to the manufacturer's protocol. By sequencing, a phagemid with an insert containing a full-length cDNA encoding an At-Rac1 homologue was identified. The sequence of this cDNA was deposited in the database and the encoded protein was designated At-Rac2. The MegAlign software package (DNASTAR Inc.) and the Clustal method were used to align the amino acid sequences of At-Rac2 and of related proteins. An A . thaliana genomic library was screened with At-Rac1 and At-Rac2 cDNA probes. Genomic fragments that specifically hybridized with each probe were subcloned and partially sequenced. Clones containing sequences identical to At-Rac1 and At-Rac2 coding sequences were identified. These clones were later found to overlap with BAC clones sequenced as part of the ESSAII project. Clones M4E13 and T19K4 contain the genomic sequences of At-Rac1 or At-Rac2 , respectively. pUCAP-GUS was constructed by cloning an expression cassette consisting of the 35S promoter, a GUS (β-glucuronidase) coding sequence interrupted by an intron ( GUS-intron ), and the nos poly A addition signal as an SalI fragment from pTJK136 into pUCAP . Sequences upstream of the At-Rac1 and At-Rac2 coding regions were amplified by PCR from genomic clones using a polymerase with proofreading capability ( Pfu polymerase; Stratagene). Primers were designed to amplify the entire At-Rac1 (∼1.1 kb) or At-Rac2 (∼1.9 kb) promoter regions from the ends of adjacent open reading frames (ORFs) to the beginning of the coding sequences and to introduce NcoI sites at the start codons. Amplified At-Rac promoters were fused via the NcoI site to the GUS-intron sequence by cloning restricted PCR fragments into pUCAP-GUS. Insertion of the At-Rac promoters replaced the 35S promoter originally present in this construct. The resulting plasmids were used to analyze promoter activity in growing pollen tubes in transient expression experiments. At-Rac1::GUS-intron::nos and At-Rac2::GUS-intron::nos expression cassettes were subcloned from these constructs as HindIII or XbaI fragments, respectively, into the binary vector pPZP211 . Resulting plasmids were used to generate stably transformed A . thaliana lines. Sequences encoding At-Rac2 and At-Rac2ΔCSIL were amplified by PCR from the At-Rac2 cDNA in pBluescriptSK − . A DNA fragment encoding the NH 2 -terminal catalytic domain of Clostridium difficile toxin B (TcdB; amino acids 1–546) was also amplified by PCR using the construct “CDB1-546 in pGEX-2T” as a template. Primers were designed to create an ATG context optimal for gene expression in dicot plants and to introduce stop codons at the 3′ ends of the At-Rac2 Δ CSIL and TcdB sequences. PCR fragments, after confirmation of error-free amplification by sequencing, as well as a GUS cDNA (derived from pBI121; CLONTECH Laboratories Inc.) were inserted into the pUCAP-based expression vector pLAT52MCS between the lat52 promoter and the nos poly A addition signal. QuikChange™ (Stratagene) PCR-based mutagenesis was used to create mutant sequences encoding G 15 V-At-Rac2, T 20 N-At-Rac2, and Q 64 E-At-Rac2. After sequence confirmation, mutated At-Rac2 cDNAs were subcloned into nonamplified pLAT52MCS. The cloning of sequences encoding green fluorescent protein (GFP) and GFP fused to the NH 2 terminus of the mouse talin f-actin binding domain into pLAT52MCS was described earlier . Into the same expression vector, sequences were inserted that encoded GFP fused to the NH 2 terminus of the following polypeptides: At-Rac2, At-Rac2ΔCSIL, and G 15 V-At-Rac2 (sequences subcloned from the vectors described above); the PH-domain of human PLC-δ 1 ; and the PH-domain of ADP-ribosylation factor nucleotide-binding site opener (ARNO) . The GFP-PLC-δ 1 -PH sequence in pLAT52MCS was altered by QuikChange™ mutagenesis to sequences encoding GFP-PLC-δ 1 -PH-K 32 L and GFP-PLC-δ 1 -PH-K 32 E. Correct amplification of the regions encoding mutated versions of PLC-δ 1 -PH was confirmed by sequencing. Transgenic A . thaliana ecotype Landsberg erecta lines were generated as described in Kost et al. using the Agrobacterium vacuum-infiltration method developed by Bechtold et al. . T1 seeds were plated on MS medium containing 50 mg/liter kanamycin and 100 mg/liter cefotaxime. In vitro grown, kanamycin-resistant T1 plants at different developmental stages as well as pollen collected from such plants were analyzed for GUS expression as described below. For transient expression, genes were transferred into cultured tobacco pollen tubes by particle bombardment as described in Kost et al. . Coexpression of two genes was achieved by coating particles with equal amounts of each expression vector. To assay for GUS activity, A . thaliana plants and pollen grains were immersed for 12–18 h in a X-Gluc substrate solution. Assayed plants were extracted with ethanol to remove chlorophyll and improve visibility of the blue GUS reaction product. Destained plants were examined under a dissection microscope. Plant organs and pollen grains were mounted in water between slides and coverslips for observation at higher magnifications. To visualize transient GUS expression, X-Gluc solution (1 ml per plate) was evenly distributed on the surface of solid culture medium covered with pollen tubes grown from bombarded grains. After 2 h of incubation, squares of solid medium were cut out and flipped upside-down (pollen tubes in direct contact with the glass) onto coverslips. Stained plant organs, pollen grains, and pollen tubes were analyzed by bright-field transmitted light microscopy using an Axioscope ( Carl Zeiss Inc. ) microscope. Images were taken by 35-mm photography (64T film; Eastman Kodak Co. ). Incubation in X-Gluc solution was performed at 37°C. All X-Gluc solutions were buffered with 0.1 M sodium phosphate at pH 7.0. Plants were incubated in a solution containing 0.2% X-Gluc (Jersey Lab and Gloves Supply), 0.1% Triton X-100, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 6% N , N -dimethyl-formamide (DMF). The same solution supplemented with 5% mannitol was used for pollen tubes. Pollen grains were assayed in 0.1% X-Gluc, 0.1% Triton X-100, 3% DMF, and 5% mannitol. Epifluorescent images of GFP-expressing pollen tubes were taken through a 4× lens using a standard FITC filter set and an Axioscope microscope equipped with a 100-W mercury lamp. All images were obtained by exposing 400 ASA 35 mm film (EliteII; Eastman Kodak Co. ) for 10–15 s. Confocal analysis of GFP expression was performed using an LSM410 inverted confocal microscope ( Carl Zeiss Inc. ) as outlined in Kost et al. . Methods used to determine pollen tube growth rates and techniques used for digital image processing are described in the same report. Intensity plots were created using the Scion Image software package (Scion Corp.). Recombinant glutathione S-transferase (GST) and GST fusion proteins were prepared as described by Lemichez et al. with some modifications. Synthesis of recombinant protein in Escherichia coli BL21 was induced by treatment with 0.5 mM IPTG for 2 h at 30°C. Cells were lysed and recombinant proteins were purified in PB buffer (50 mM Tris-HCl at pH 7.4; 250 mM NaCl, 5 mM MgCl 2 , 0.1 mM DTT). After purification, recombinant proteins on agarose beads were resuspended in PB buffer containing 50% glycerol, frozen in liquid nitrogen, and stored at −80°C. For GTP binding and kinase binding assays, recombinant proteins on agarose beads were loaded with nucleotides for 20 min at room temperature in loading buffer (50 mM Tris-HCl at pH 7.4; 25 mM NaCl, 1 mM EDTA, 0.5 mM DTT) containing 0.1 mM [γ- 32 P]GTP (6,000 Ci/mmol; New England Nuclear ), GTPγS, or GDPβS (both Boehringer Mannheim ), followed by addition of MgCl 2 to a final concentration of 20 mM. GTP binding and GTPase activity assays were performed using protocols established by Self and Hall . Tobacco pollen tubes (1 g fresh weight) grown for 3 h in liquid PT medium were washed twice with a solution containing 150 mM potassium phosphate (pH 7.2), 0.4 M mannitol, and 150 mM NaCl. After resuspension in 5 ml lysis buffer, pollen tubes were passed twice through a Dounce homogenizer. If nothing else is indicated, a solution containing 10 mM Hepes (pH 7.4) and 0.8 M sorbitol was used as lysis buffer. All lysis buffers used were supplemented with a protease inhibitor cocktail (Complete™, EDTA-free; Boehringer Mannheim ) according to the manufacturer's instructions. Polyclonal anti–At-Rac antiserum was obtained from rabbits immunogenized with GST-At-Rac1. Standard methods described in Harlow and Lane were used to affinity purify specific anti–At-Rac antibodies (α-At-Rac) on nitrocellulose membranes loaded by blotting with thrombin-treated, GST-free At-Rac1. Pollen tube and recombinant proteins were resolved by SDS-PAGE according to Laemmli under reducing conditions using 12% gels. For immunoblotting, proteins were transferred onto PVDF membranes (Schleicher & Schuell). Membranes were incubated with primary antibodies, affinity-purified α-At-Rac (diluted 1:200) or a monoclonal mouse anti-actin antibody ( Boehringer Mannheim ; diluted 1:2,500), and secondary antibodies, peroxidase-conjugated goat anti–rabbit or goat anti–mouse antibodies ( Boehringer Mannheim ; diluted 1:5,000), for 1 h at room temperature in Tris-buffered saline (TBS; 25 mM Tris-HCl at pH 7.4; 137 mM NaCl, 5 mM KCl) supplemented with 4% BSA . After incubation with primary and secondary antibodies, membranes were washed twice for 10 min with TBS containing 4% BSA and 0.02% Tween 20. After the final wash, membrane-associated peroxidase activity was visualized using the ECL kit ( Boehringer Mannheim ) according to the manufacturer's instructions. Pollen tube extracts were prepared as described above with homogenization performed in FB (50 mM triethanolamine at pH 7.8; 150 mM KCl, 1 mM DTT, 5 mM MgCl 2 , protease inhibitors). TcdB glucosylation reactions were carried out as described in Just et al. . In brief, 0.5 μg TcdB (obtained from Ingo Just, Albert-Ludwigs-Universität, Freiburg, Germany) and 0.3 μCi UDP-[ 14 C]-glucose (300 mCi/mmol; New England Nuclear ) in FB buffer were added to extract containing 10 μg total pollen protein or to PB containing 1 μg recombinant GST-At-Rac2 to obtain a final volume of 20 μl. The reaction mix was scaled up to a total volume of 200 μl for immunoprecipitation experiments. Reactions were carried out at 37°C for 1 h. Products were resolved by SDS-PAGE either directly or after immunoprecipitation with affinity-purified α-At-Rac (diluted 1:20). Gels were incubated for 10 min in 1 M salicylic acid before drying. Pollen tube extracts were centrifuged for 10 min at 10,000 g to obtain a post-nuclear supernatant (PNS) with a protein concentration of ∼0.25 mg/ml. The resulting PNS was centrifuged at 100,000 g for 1 h (SW-55 Ti rotor; Beckman Instruments Inc. ) to separate cytoplasmic (supernatant) and membrane (pellet) fractions. Pelleted membranes were resuspended in lysis buffer. Proteins contained in 20 μl of each PNS, cytoplasmic, and resuspended membrane fractions were analyzed by immunoblotting. TcdB glucosylation assays were carried out as described above using 5 μl of each fraction. Kinase binding assays were performed as described by Ren et al. with some modifications. Pollen tube extracts were prepared as described above with homogenization performed in buffer A (50 mM Tris-HCl at pH 7.4; 1% Triton X-100, 100 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, protease inhibitors). Extracts were clarified by centrifugation at 13,000 g for 15 min at 4°C. Agarose beads bound to GST (25 μg protein) or to GST-At-Rac2 (2.5 μg protein) loaded with nucleotides as described above were added to 500 μl clarified extract (0.5 mg/ml total protein) and incubated for 2 h at 4°C. After incubation, beads were washed three times in buffer B (50 mM Tris-HCl at pH 7.4; 0.1% Triton X-100, 50 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM DTT) and once in buffer C (50 mM Tris-HCl at pH 7.4; 0.02% Triton X-100, 100 mM NaCl, 5 mM MgCl 2 , 0.1 mM EGTA, 1 mM DTT). After resuspension in buffer C during the last washing step, one-tenth of the total volume of each sample was analyzed by SDS-PAGE and Coomassie brilliant blue staining to verify concentration and quality of the recombinant proteins used. Washed beads were resuspended in 35 μl buffer C. Samples used to investigate effects of phosphatidic acid on At-Rac2–associated kinase activity were treated similarly, but Triton X-100 was omitted from all buffers except for buffer A. Beads in 35 μl buffer C were supplemented with 20 μg lipid substrates in 10 μl 10 mM Tris-HCl, pH 7.4, and incubated at room temperature for 10 min. Either PtdIns 4-P or a mix of phosphoinositides was used as a substrate. Some samples were supplemented with phosphatidic acid to a final concentration of 40 μM. Kinase reactions were started by adding 5 μl ATP buffer (50 mM Tris-HCl at pH 7.4; 5 mM MgCl 2 , 0.5 mM ATP, 10–25 μCi [γ- 32 P]ATP, 6,000 Ci/mmol; New England Nuclear ) to each sample. After 7 min, reactions were stopped with 80 μl HCl (1 N). Lipids were extracted in 160 μl methanol/chloroform (1:1) by vigorous mixing for 1 min. Organic and aqueous phases were separated by centrifugation. The lower organic phase was washed with 260 μl HCl (1 N)/methanol/chloroform (48:47:3), concentrated under N 2 to a volume of 20 μl and spotted on Oxalate-EDTA–impregnated silica gel plates (LK6D; Whatman Inc.). Chromatography was performed as described by Pignataro and Ascoli . Radioactivity emitted from membranes, gels, or TLC plates was visualized using a PhosphorImager detection system (Molecular Dynamics). 32 P-labeled products of kinase binding assays performed as described above using a mix of phosphoinositides as lipid substrate were deacylated, mixed with 3 H-labeled standards, and analyzed by anion-exchange HPLC using a Partisphere SAX column (Whatman) as described by Serunian et al. . Elution of assay products and standards was detected simultaneously using an on-line continuous flow scintillation detector (Radiomatic ® FSA; Packard Instrument Co. ). Previously, we reported the cloning of At-Rac1 , which was identified in a screen for A . thaliana cDNAs that cause morphological changes when expressed in yeast . Using At-Rac1 as a probe, At-Rac2 was isolated from an A . thaliana cDNA library. The corresponding genomic sequences were cloned by screening an A . thaliana library with At-Rac1 or At-Rac2 cDNA probes. Genomic sequences upstream of the At-Rac1 and At-Rac2 ORFs, including the ends of adjacent ORFs and the entire At-Rac 5′ untranslated regions, were fused to a cDNA encoding GUS . Transgenic A . thaliana lines containing the resulting At-Rac1 or At-Rac2 promoter- GUS fusion constructs ( At-Rac1::GUS , At-Rac2::GUS ) were generated and histochemically analyzed for GUS expression. Four independent lines transformed with At-Rac2::GUS showed GUS expression confined to pollen , to growing pollen tubes, to stipules, and to a small region of the vascular tissue below the cotyledons (data not shown). In contrast, six lines transformed with At-Rac1::GUS were all found to express GUS in the vascular tissues of all organs. Only one of these lines showed a weak GUS expression in pollen (data not shown). GUS is known to be a stable protein with a slow turnover rate in plant cells . To confirm that GUS activity detected in transgenic pollen tubes resulted at least partially from gene expression during pollen germination and tube growth, promoter- GUS fusion constructs were also transiently expressed in growing tobacco ( Nicotiana tabacum ) pollen tubes. Under these conditions, the At-Rac2 promoter was found to confer strong GUS expression at a level similar to that obtained with the pollen-specific lat52 promoter . No GUS expression was observed in pollen tubes transiently transfected with At-Rac1::GUS or with a control construct containing a cDNA encoding GFP fused to the lat52 promoter . These results indicate that At-Rac2 , but not At-Rac1 , is preferentially expressed in pollen and in growing pollen tubes. At-Rac1 and At-Rac2 encode proteins of 21 kD that show high homology to human HsRac1 . Recombinant At-Rac1 and At-Rac2 were both found to bind and to hydrolyze GTP (data not shown), confirming that the two proteins are indeed functional small GTPases. They belong to a large family of Rac-like proteins with unknown or poorly characterized cellular functions whose genes have been cloned recently from different plant species. The protein sequences of Arac1 and Rop1At , two A . thaliana Rac-like proteins that share very high sequence homology with At-Rac2 (98.5 and 95.4% identical amino acids, respectively), are shown in Fig. 2 . Interestingly, Rop1At has been shown to be expressed in pollen and was proposed to have a function in the regulation of pollen tube growth . We found that in situ analysis of At-Rac2 function in cultured A . thaliana pollen tubes is not feasible. These cells elongate slowly, show severe morphological abnormalities, and cease growing only a few hours after germination . By contrast, tobacco pollen tubes can grow rapidly and morphologically normal in vitro for up to 48 h . Methods have been established that allow analysis of transient gene expression in cultured tobacco pollen tubes after gene transfer into germinating pollen grains by particle bombardment . We have used these methods, along with biochemical techniques, to study the function of At-Rac2 and its tobacco homologues in the regulation of pollen tube growth. An antibody raised against At-Rac1 (α-At-Rac) was found to have similar affinities to recombinant At-Rac1 and At-Rac2 (data not shown). Immunoblot analysis showed that in tobacco pollen tube extracts α-At-Rac binds to at least one protein with an apparent molecular mass of 21 kD , which corresponds to the size of Rac. Clostridium difficile toxin B (TcdB) is known to glucosylate and thereby inactivate specifically mammalian Rho family proteins including Rac . Treatment with TcdB and UDP-[ 14 C]-glucose resulted in glucosylation and labeling of recombinant At-Rac2 (data not shown), showing that plant Rac homologues can serve as substrates of this toxin. Radiolabeled proteins of 21 kD were detected in tobacco pollen tube extracts assayed for glucosylation by TcdB, but not in control samples treated with UDP-[ 14 C]-glucose alone . To investigate whether the detected pollen tube TcdB targets are recognized by α-At-Rac, proteins interacting with this antibody were immunoprecipitated from tobacco pollen tube extracts assayed for glucosylation by TcdB. In these experiments, at least one 21-kD pollen tube protein glucosylated by TcdB was found to interact with α-At-Rac and was therefore identified as an At-Rac2 homologue. No radiolabeled proteins were precipitated from assayed extracts treated with preimmune serum . A cDNA encoding the catalytic domain of TcdB was transiently expressed under the control of the lat52 promoter in tobacco pollen tubes. Fluorescence emitted by coexpressed GFP was used to identify successfully targeted pollen tubes among an excess of untransformed, nonfluorescent tubes. Transient expression of marker genes did not affect pollen tube growth. Control pollen tubes transfected with expression vectors containing GFP and GUS coding sequences fused to the lat52 promoter showed normal morphology and elongated at the same rate as untransformed pollen tubes (data not shown). By contrast, transient expression of the catalytic domain of TcdB clearly inhibited tobacco pollen tube growth . Together, these results indicate that tobacco pollen tubes contain at least one At-Rac2 homologue with an essential function in tube elongation. The function and the intracellular localization of Ras related small GTPases can be predictably altered by introducing specific mutations in conserved domains . Sequences encoding wild-type and mutant forms of At-Rac2 were transiently expressed in growing tobacco pollen tubes under the control of the lat52 promoter. Coexpression of GFP was used to identify targeted tubes. Expression of dominant negative T 20 N-At-Rac2 strongly inhibited pollen tube extension . After reaching a total length (tip to grain) of several 100 μm, T 20 N-At-Rac2–expressing pollen tubes stopped elongating. By contrast, expression of constitutive active G 15 V-At-Rac2 or Q 64 E-At-Rac2 caused germinating pollen tubes to form large, spherical balloons instead of elongating tubes . Apparently, a complete loss of polarity of cell extension was induced. Wild-type At-Rac2 had similar although somewhat weaker effects . After germination, pollen tubes elongated normally for a short while before tips started to form balloons. The balloons often had irregular shapes instead of being spherical, presumably because the potential for polarized growth was not completely abolished. At-Rac2ΔCSIL, lacking the COOH-terminal consensus prenylation domain known to be required for the membrane association of Ras related proteins, still induced depolarized tip growth but was clearly less effective than wild-type At-Rac2 . These results demonstrate a key role of At-Rac2 homologous proteins in the regulation of polar pollen tube growth and indicate that membrane localization is essential for their activity. The best characterized function of Rho family proteins in animal and yeast cells is the regulation of actin organization . Therefore, effects of transient expression of mutant At-Rac2 on the tobacco pollen tube actin cytoskeleton were examined. G 15 V-At-Rac2 or T 20 N-At-Rac2 were coexpressed in pollen tubes with a GFP-mouse talin fusion protein, which we have shown to label plant actin filaments in vivo in a specific and noninvasive manner . The actin cytoskeleton in normally growing tobacco pollen tubes consists of thick, longitudinally oriented actin bundles in the shank and fine filamentous structures close to the tip . An excessive number of thick actin cables arranged in a helical pattern was observed in balloons formed by G 15 V-At-Rac2–expressing pollen tubes . Expression of constitutive active At-Rac2 apparently induced actin polymerization or bundling and, possibly, reorientation of actin cables in the pollen tube tip. By contrast, actin bundles in pollen tubes expressing dominant negative T 20 N-At-Rac2 were generally finer and less organized than in control tubes, indicating that inhibition of pollen tube Rac activity may reduce actin bundling . As in other cell types, one of the functions of pollen tube Rac appears to be the regulation of the actin cytoskeleton. Analysis of pollen tube extracts revealed that a significant portion of the 21-kD tobacco pollen tube protein that interacts with the α-At-Rac antibody cofractionates with membranes, whereas the majority of the protein remains in the cytoplasmic fraction . A similar distribution of radiolabeled protein with the same size was observed when pollen tube extracts assayed for glucosylation by TcdB were fractionated . In control experiments, pollen tube actin showed a typical distribution between cytoplasmic and membrane fractions . To investigate the intracellular localization of pollen tube Rac in more detail, sequences encoding wild-type and mutant forms of At-Rac2 fused to the COOH terminus of GFP were transiently expressed in tobacco pollen tubes under the control of the lat52 promoter. A GFP cDNA was also expressed from the same promoter. Although somewhat weaker, effects of expressing the fusion proteins on pollen tube growth were very similar to those observed with untagged wild-type or mutant At-Rac2 . This indicates that the fusion proteins were functional and that analysis of their localization provided relevant information on the intracellular distribution of pollen tube Rac proteins. Tobacco pollen tubes expressing GFP or GFP fused to different forms of At-Rac2 were examined by confocal microscopy. GFP was found to be evenly distributed in the pollen tube cytoplasm . Confocal optical sections through pollen tubes expressing GFP fused to constitutive active or to wild-type At-Rac2 revealed that these fusion proteins accumulated at the cell cortex exclusively in the tube tip, indicating that they associated with the plasma membrane specifically in this pollen tube region. The rest of the fusion proteins was evenly distributed in the cytoplasm . GFP fused to At-Rac2ΔCSIL did not show membrane association and was localized to the cytoplasm similar to GFP . In summary, the results described here indicate that At-Rac2 homologues localize specifically to the plasma membrane at the pollen tube tip and confirm that the COOH-terminal prenylation domain is essential for their proper localization and function. PtdIns P-Ks and PtdIns 4, 5-P 2 were identified as effectors of mammalian Rho family small GTPases . Rac and Rho, both in the GTP- and in the GDP-bound conformation, have been demonstrated to physically interact with PtdIns P-K activity in mammalian cell extracts . We have tested the possibility that PtdIns P-Ks and PtdIns 4, 5-P 2 act as Rac effectors also in pollen tubes. Experiments were performed to investigate whether At-Rac2 and its tobacco homologues bind to PtdIns P-K activity present in tobacco pollen tube extracts. Recombinant At-Rac2 fused to the COOH terminus of GST was loaded with the nonhydrolyzable nucleotide analogues GTPγS or GDPβS. Proteins interacting with nucleotide-bound GST-At-Rac2 were purified from tobacco pollen tube extracts and assayed for lipid kinase activity using [γ- 32 P]ATP and a mix of phosphoinositides as substrates. An excess of GST was used in control experiments. Labeled lipids produced in kinase assays were analyzed by TLC and autoradiography. Fig. 8 a (top) shows that significant amounts of radiolabeled PtdIns P 2 were synthesized when proteins associated with GTPγS- or GDPβS-loaded GST-At-Rac2 were assayed, whereas minimal levels of PtdIns P 2 production were detected in GST control samples. Similar results were obtained when purified PtdIns 4-P was used as a substrate instead of a mixture of phosphoinositides or when proteins coimmunoprecipitated with At-Rac2 homologues from tobacco pollen tube extracts by the α-At-Rac antibody were assayed (data not shown). The different PtdIns P 2 isoforms identified to date in plant and/or animal cells, including PtdIns 4, 5-P 2 , PtdIns 3, 4-P 2 and PtdIns 3, 5-P 2 , are difficult to separate on TLC plates. Therefore, 32 P-labeled lipid products of kinase binding assay performed as described above using a mix of phosphoinositides as substrates were deacylated and subjected to HPLC. Under conditions that allow clear separation of deacylated PtdIns P 2 isoforms , 32 P-labeled, deacylated assay products coeluted from HPLC columns with 3 H-labeled GroPIns 4, 5-P 2 used as a standard . The described results demonstrate that recombinant At-Rac2, both in the GTP- and in the GDP-bound conformation, as well as at least one of its tobacco homologues, physically associates with tobacco pollen tube PtdIns P-K activity, which synthesizes specifically PtdIns 4, 5-P 2 . The isolated PH-domain of PLC-δ 1 has been shown to bind PtdIns 4, 5-P 2 integrated into lipid membranes specifically and with high affinity, both in vitro and in vivo . Under the control of the lat52 promoter, a sequence encoding a PLC-δ 1 PH-domain GFP fusion protein (GFP-PLC-δ 1 -PH) was transiently expressed in tobacco pollen tubes. Consistent with a key role of PtdIns 4, 5-P 2 in the regulation of pollen tube growth, moderate levels of GFP-PLC-δ 1 -PH expression were sufficient to strongly inhibit tobacco pollen germination and tube growth . Only weakly fluorescent GFP-PLC-δ 1 -PH–expressing pollen tubes were able to grow normally. Fluorescence emitted by these pollen tubes was too weak to be visible on micrographs like the one shown in Fig. 9 a. Confocal optical sections through such pollen tubes revealed that GFP-PLC-δ 1 -PH accumulated at the plasma membrane in the tube tip . Although GFP-PLC-δ 1 -PH appeared to label a somewhat smaller area, its localization in tobacco pollen tubes was strikingly similar to that of transiently expressed GFP-At-Rac2 fusion proteins . Pollen tubes with GFP-PLC-δ 1 -PH labeling as shown in Fig. 9 e were indistinguishable from untransformed control tubes in terms of morphology and growth rate (the average growth rate of 20 weakly fluorescent, GFP-PLC-δ 1 -PH– expressing pollen tubes was 5 μm/s, which is identical to the average growth rate of untransformed pollen tubes), demonstrating that the observed localization of the fusion protein provides information on a physiologically normal situation. A number of control experiments supported the view that the specific interaction of GFP-PLC-δ 1 -PH with PtdIns 4, 5-P 2 caused inhibition of pollen tube growth and was responsible for the localization of the fusion protein to the plasma membrane at the tip. The free amino group of the lysine residue at position 32 in the PLC-δ 1 PH-domain has been shown to form a direct hydrogen bond to the 4-phosphate of PtdIns 4, 5-P 2 . Replacement of this basic lysine residue by neutral leucine (K 32 L) or even by glutamic acid (K 32 E) was demonstrated to abolish the ability of PLC-δ 1 to bind PtdIns 4, 5-P 2 in membranes . Neither GFP-PLC-δ 1 -PH-K 32 L nor GFP-PLC-δ 1 -PH-K 32 E detectably accumulated at the tip membrane when transiently expressed in tobacco pollen tubes. Whereas high level expression of GFP-PLC-δ 1 -PH-K 32 L still severely inhibited pollen tube growth , even brightly fluorescent tubes expressing GFP-PLC-δ 1 -PH-K 32 E could elongate rapidly and showed an essentially normal morphology . The PH-domain of ARNO has been demonstrated to bind with a high degree of specificity to PtdIns 3, 4, 5-P 3 , a lipid that has not been identified in plant cells to date . Transient expression from the lat52 promoter of a cDNA sequence encoding the ARNO PH-domain fused to GFP did not significantly affect tobacco pollen tube growth . No accumulation of the GFP-ARNO-PH fusion protein at the plasma membrane was observed . These results provide strong evidence for an essential function of PtdIns 4, 5-P 2 in pollen tube elongation. Rac proteins, which physically interact with PtdIns 4, 5-P 2 synthesizing PtdIns P-K activity, and PtdIns 4, 5-P 2 both localize to the plasma membrane at the pollen tube tip. This suggests that Rac proteins, PtdIns P-K activity, and PtdIns 4, 5-P 2 act together in a common pathway to regulate polar pollen tube growth. Transient expression of wild-type or constitutive active At-Rac2 in tobacco pollen tubes resulted in the formation of large balloons instead of elongated tips. The cell wall of pollen tubes, similar to that of most plant cells, must withstand turgor pressure built up in the protoplast by osmotic water uptake. Certain treatments, including incubation in hypotonic media, can induce some swelling of pollen tubes at the tip, where the newly formed cell wall is relatively elastic and has not yet attained its ultimate rigidity. However, pollen tube tips generally burst before the swelling induced by such treatments results in a substantial increase of their diameter . Extensive balloon formation as induced by Rac overexpression requires the formation of rigid cell wall structures which depends on constant deposition of new cell wall material. Balloons induced by Rac overexpression clearly resulted from organized but depolarized growth. Whereas Rac overexpression depolarized pollen tube extension, inhibition of endogenous Rac activity by transient expression of a dominant negative At-Rac2 or of TcdB completely inhibited the growth of these cells. These observations demonstrate that pollen tube Rac is essential for growth and plays a key role in the determination of growth polarity. A pea pollen tube Rac homologue was determined to be localized to the plasma membrane at the tip using immunofluorescence techniques . Chemical fixation and permeabilization required for such experiments are known to severely change the structure of pollen tube cells and may affect Rac localization. We have chosen to use GFP as a tag to investigate the intracellular localization of At-Rac2 in living pollen tubes. This technique has been successfully employed to analyze the intracellular distribution of related small GTPases in different cell types . Transient expression of GFP-At-Rac2 fusion proteins and of corresponding untagged At-Rac proteins had similar effects on pollen tube growth. This demonstrates that the fusion proteins were functional and valid indicators of At-Rac2 localization. A few hours after particle bombardment, when effects on pollen tube morphology started to become apparent, GFP-At-Rac2 fusion proteins were associated with an extended area of the plasma membrane at the tip. At this stage, their localization was very similar to the intracellular distribution of pea pollen tube Rac as observed by immunofluorescence. At later stages, the fusion proteins localized to the plasma membrane throughout the balloons formed (Kost, B., and N.-H. Chua, unpublished observations). Pollen tubes transiently transformed with GFP-At-Rac2 sequences emitted fluorescence of varying intensities, indicating that they expressed the fusion proteins at different levels. Using a truncated lat52 promoter or the 35S promoter, which both confer lower expression in pollen tubes as compared with the full-length lat52 promoter , resulted in a reduction of the total number of pollen tubes emitting detectable fluorescence. However, independent of the promoter used, all fluorescent pollen tubes analyzed displayed depolarized growth and localization of GFP-At-Rac2 fusion proteins as described above (Kost, B., and N.-H. Chua, unpublished observations). This indicates that expression of the fusion proteins at minimal levels required for visualization by fluorescence microscopy is sufficient to affect pollen tube growth. Because fluorescence detection may require relatively high concentrations of GFP fusion proteins, it is possible that endogenous Rac in normally growing pollen tubes localizes to a more restricted area of the tip plasma membrane as compared with GFP-At-Rac2 fusion proteins in transiently transformed tubes. Additional experiments, e.g., immunogold labeling of sections through physically fixed pollen tubes, may be required to determine the exact extension of the plasma membrane area that is Rac associated in normally growing pollen tubes. Nevertheless, our results and the earlier immunolocalization study clearly demonstrate that Rac localizes to pollen tube plasma membrane specifically in the tip region. In normally elongating pollen tubes, the actin cytoskeleton consists essentially of longitudinally oriented thick actin cables that mediate cytoplasmic streaming and of fine actin structures in the tube apex that may have a direct function in polarized secretion . Transient expression of mutant Rac was found to alter pollen tube actin organization. The clearest effect observed was the formation of extensive actin cables in growing balloons induced by expression of constitutive active Rac. Expression of dominant negative Rac resulted in a reduction of actin bundling. Interfering with the activity of Rho type small GTPases in fibroblasts is known to have comparable effects. In these cells, Rho activation leads to the formation of actin cables, whereas its inactivation results in the disappearance of thick actin bundles . As its animal and yeast homologues, pollen tube Rac functions in the regulation of actin organization. However, the observed effects on the actin cytoskeleton alone are unlikely to account for the dramatic changes in pollen tube growth induced by the expression of mutant Rac. Pollen tube elongation is thought to be based on polarized secretion restricted to the apex . As suggested for some of its animal and yeast homologues, pollen tube Rac may control directed secretion possibly via the coordinated regulation of actin organization and of exocytotic membrane traffic. In normally elongating pollen tubes, activated endogenous Rac associated with the plasma membrane in a restricted area at the tip may organize directed secretion to this site. Inactivation of endogenous Rac by transient expression of TcdB or of dominant negative mutant Rac was found to inhibit pollen tube growth, presumably by blocking secretion. By contrast, expression of constitutive active Rac led to the formation of balloons instead of elongated tips, conceivably because it resulted in an extension of the membrane area associated with activated Rac, which caused depolarized secretion and growth. Overexpressed wild-type Rac was apparently partially activated by endogenous factors and had similar, although somewhat weaker, effects. Deletion of the COOH-terminal CAAX-domain clearly reduced, but did not complete abolish, the potential of wild-type Rac to induce depolarized growth. Even in the absence of a membrane targeting domain, local concentrations of At-Rac2ΔCSIL at the tip plasma membrane achieved by transient expression of a sequence encoding this protein under the control of the strong lat52 promoter were apparently high enough for some stimulation of ectopic secretion. Estimations based on simple geometric calculations revealed that the total surface of pollen tubes transiently expressing constitutive active Rac was about four times smaller than that of control pollen tubes at the time of analysis (12–18 h after particle bombardment). Whereas this appears to be in contradiction with a role of activated Rac in the stimulation of secretion, it likely results from the disruption of cytoplasmic organization caused by transient expression of constitutive active Rac. The total volume of the pollen tube cytoplasm, which remains essentially constant after pollen germination, was estimated to be ∼20 times smaller than the volume of balloons formed by constitutive active At-Rac2–expressing pollen tubes. As a consequence, the cytoplasm could only form a thin layer at the inner surface of these balloons, with the remainder of the volume filled by large vacuoles. It is conceivable that the proceeding drastic disruption of cytoplasmic organization during balloon formation increasingly interfered with the efficient delivery of secretory vesicles to the plasma membrane. Mammalian Rho family small GTPases in the GTP-bound conformation have been found to stimulate PtdIns P-K activity and synthesis of PtdIns 4, 5-P 2 in permeabilized cells and in cell lysates . Recombinant as well as endogenously produced mammalian Rac and Rho were shown to physically interact with a PtdIns P-K activity in cell extracts . Here, we present compelling evidence that pollen tube Rac, PtdIns P-K, and PtdIns 4, 5-P 2 cooperate in a common pathway to regulate polar pollen tube growth. Our results indicate that PtdIns P-K and PtdIns 4, 5-P 2 may act as Rac effectors in pollen tubes, as they do in mammalian cells. Rac inactivation by transient expression of dominant negative mutant forms of this protein inhibited pollen tube growth. The same effect was observed when the interaction of PtdIns 4, 5-P 2 with its downstream targets was disrupted by transient expression of GFP-PLC-δ 1 -PH, which binds strongly and specifically to this lipid. Recombinant At-Rac2 and its endogenous tobacco homologues were demonstrated to physically associate in pollen tube extracts with PtdIns P-K activity that synthesizes specifically PtdIns 4, 5-P 2 . Rac and PtdIns 4, 5-P 2 were both observed to localize to the plasma membrane specifically at the pollen tube tip. Interestingly, recombinant mammalian Rac and Rho as well as pollen tube At-Rac2 were found to interact with a PtdIns P-K activity both in the activated GTP-bound and in the inactive GDP-bound form. In a recent report, evidence was presented indicating that the COOH-terminal end of mammalian Rac is mainly responsible for the interaction of this protein with PtdIns P-K and not the NH 2 -terminally localized effector domain, which is known to undergo drastic conformational changes upon GTP binding . As was suggested for its mammalian homologues, activated pollen tube Rac may stimulate PtdIns P-K activity and PtdIns 4, 5-P 2 synthesis via GTP-dependent binding of an additional, unidentified cofactor or by translocating a constitutively associated PtdIns P-K from the cytoplasm to the plasma membrane . In vivo and in vitro experiments with mutant At-Rac2 are currently being performed to further characterize the interaction between Rac and PtdIns P-K activity in pollen tubes. Recent results have established that two different PtdIns P-K–dependent pathways contribute to the synthesis of PtdIns 4, 5-P 2 in mammalian cells. In addition to phosphorylation of PtdIns 4-P by PtdIns 4-P 5-K, which represents a well established pathway, phosphorylation of PtdIns 5-P at position 4 of the inositol ring was found to be catalyzed by PtdIns 5-P 4-Ks, formerly known as type II PtdIns 4-P 5-K . Rac-associated pollen tube lipid kinase activity generated PtdIns 4, 5-P 2 when PtdIns 4-P was used as a substrate. Because commercially available PtdIns 4-P preparations may contain traces of PtdIns 5-P , this does not entirely rule out the possibility that pollen tube Rac interacts with PtdIns 5-P 4-Ks activity. However, the activity of the Rac-associated pollen tube lipid kinase could be stimulated by phosphatidic acid (Lemichez, E., and N.-H. Chua, unpublished observation), which is considered to be characteristic for PtdIns 4-P 5-Ks in mammalian systems . Therefore, it appears likely that we have detected an interaction between Rac and PtdIns 4-P 5-Ks activity in pollen tubes. Signaling appears to often involve recruitment of regulatory proteins to specific membrane domains where these proteins form complexes that organize local cellular responses. A large number of regulatory proteins is known to bind PtdIns 4, 5-P 2 or other phosphoinositides specifically and with high affinity. This has led to the idea that localized phosphoinositide synthesis may have a key function in spatially restricted signaling events. However, only limited evidence for lipid compartmentalization in cells has been generated to date and the mechanisms involved in the regulation of localized synthesis of particular membrane lipids are unknown . Our results provide direct evidence showing that PtdIns 4, 5-P 2 accumulates in the plasma membrane of living pollen tubes specifically at the tip and indicate that the observed PtdIns 4, 5-P 2 compartmentalization is controlled by Rac homologues. By causing translocation of actin-binding proteins, tip-localized PtdIns 4, 5-P 2 may induce uncapping and bundling of actin filaments, which could stimulate elongation of longitudinally oriented actin cables in growing pollen tubes, and control the formation of actin structures present in the apical dome. In addition to its effect on actin organization, PtdIns 4, 5-P 2 may directly control exocytosis at the pollen tube tip, either by recruiting proteins that regulate membrane fusion or by locally altering membrane lipid composition. PtdIns 4, 5-P 2 potentially represents the main effector of activated Rac in pollen tubes. It may initiate the formation of complexes of regulatory proteins at the plasma membrane in the tube apex which control actin organization, targeted secretion, and polar growth. Activated Rac possibly stabilizes these complexes as well as their interaction with PtdIns 4, 5-P 2 . GFP-PLC-δ 1 -PH did not detectably accumulate at the tip plasma membrane of pollen tubes showing depolarized growth induced by coexpressed constitutive active At-Rac2 (Kost, B., and N.-H. Chua, unpublished observations). In such pollen tubes, access of the GFP-PLC-δ 1 -PH fusion protein to PtdIns 4, 5-P 2 may have been blocked by tightly bound regulatory proteins. In addition to acting as an effector itself, PtdIns 4, 5-P 2 at the pollen tube tip may also function as a precursor for the generation of other signaling molecules. Hydrolysis of PtdIns 4, 5-P 2 by PLC, which results in the formation of inositol 1, 4, 5-triphosphate (Ins-P 3 ) and diacylglycerol, followed by Ins-P 3 –induced Ca 2+ release from intracellular stores, is a key element of many known signaling events . Recently published results have indicated an essential role of this pathway in poppy pollen tube elongation . PLC-mediated hydrolysis of tip localized PtdIns 4, 5-P 2 and Ins-P 3 – induced Ca 2+ influx into the cytoplasm may be involved in the establishment of the tip-focused Ca 2+ gradient, which is known to have an important function in the regulation of pollen tube growth. ER elements are present in the clear zone at the pollen tube tip and could function as Ins-P 3 –sensitive Ca 2+ stores. Alternatively, putative Ins-P 3 –regulated Ca 2+ channels, which allow Ca 2+ influx from the extracellular matrix, may be present in the pollen tube plasma membrane. Our results strongly suggest that Rac homologues act in a common pathway with a PtdIns P-K (probably PtdIns 4-P 5-K) activity and PtdIns 4, 5-P 2 to regulate polar pollen tube growth. We present direct evidence for PtdIns 4, 5-P 2 compartmentalization in the plasma membrane at the pollen tube tip, which appears to be derived from Rac-controlled local activation of a PtdIns P-K. PtdIns 4, 5-P 2 localized to the plasma membrane at the tip could potentially act as the main Rac effector in pollen tubes by directly regulating actin-mediated targeted secretion and polarized growth. It may also serve as a substrate for Ins-P 3 production by PLC activity, which could have a function in establishing the tip-focused Ca 2+ gradient known to be involved in the regulation of pollen tube elongation . | Study | biomedical | en | 0.999997 |
10209028 | MTLn3 metastatic rat mammary adenocarcinoma cells were provided by Dr. Garth Nicholson (M.D. Anderson Cancer Center, Houston, TX). Cells were grown in alpha-MEM ( Gibco Laboratories ), supplemented with 5% FCS and antibiotics, as previously described . Unless otherwise mentioned, MTLn3 cells were prepared for all experiments as follows: cells were plated at low density in complete medium for ∼24 h and starved for 3 h before the experiment in alpha-MEM medium supplemented with 0.35% BSA and 12 mM Hepes (starvation medium). Stimulation was done with a final concentration of 5 nM murine epidermal growth factor (EGF 1 ; Life Technologies, Inc.) in starvation medium. Monospecific anti-Arp3, anti-p21, and anti-p34 rabbit polyclonal antibodies were provided by Dr. Matthew Welch (Department of Molecular and Cell Biology, University of California, Berkeley, CA), and have been characterized previously . Anti-actin mAb was purchased from Boehringer Mannheim . Cy5-conjugated goat anti–mouse IgG was purchased from Accurate Laboratories and Scientific Corp. Fluorescein-conjugated goat anti–rabbit IgG was purchased from Cappel Laboratories. Goat antibiotin antibodies coupled to 5 nm gold particles, and goat anti–rabbit antibodies coupled to 10 nm gold particles were purchased from Nanoprobes. Cells were plated on dishes (Mattek) or coverslips as previously described and stimulated with EGF or left untreated. They were fixed for 5 min at 37°C with 3.7% formaldehyde in a buffer containing 5 mM KCl, 137 mM NaCl, 4 mM NaHCO 3 , 0.4 mM KH 2 PO 4 , 1.1 mM Na 2 HPO 4 , 2 mM MgCl 2 , 5 mM Pipes, 2 mM EGTA, and 5.5 mM glucose . They were treated with cold methanol for 2 min, rinsed, and permeabilized for 20 min at room temperature in 0.5% Triton X-100 in stabilization buffer. Next, the cells were rinsed once with 0.1 M glycine in stabilization buffer and incubated for an additional 10 min in glycine. After five washes with TBS (Tris 20 mM, NaCl 154 mM, pH 8), the preparations were blocked/stabilized by incubation for 20 min with 5 μM phalloidin ( Calbiochem-Novabiochem Corp. ) in TBS, pH 8, supplemented with 1% BSA and 1% FCS. Cells were incubated further for 1 h with primary antibodies followed by five rinses in TBS plus 1% BSA and incubation for 1 h with Cy5-conjugated anti–mouse antibodies and FITC-conjugated anti– rabbit antibodies. After final washes, the coverslips were mounted in 50% glycerol in TBS supplemented with 6 mg/ml N -propyl gallate. Nucleation sites were visualized using a previously described protocol with slight modifications. Briefly, cells grown on Mattek dishes were stimulated with EGF, and permeabilized in the presence of 0.45 μM rhodamine-labeled actin in buffer C (138 mM KCl, 10 mM Pipes, pH 6.9, 0.1 mM ATP, 3 mM EGTA, pH 6.9, 4 mM MgCl 2 ) with 0.025% saponin and 1% BSA for 1 min. After a brief rinse in buffer C cells were fixed in 3.7% formaldehyde in cytoskeleton stabilization buffer (see above) for 5 min, followed by a 10 min incubation in 0.1 M glycine in cytoskeleton stabilization buffer. After a rinse in TBS, samples were incubated with 5 μM phalloidin for 20 min in TBS/BSA/FCS (pH 8.1, see above), washed five times for 5 min with TBS/BSA, and mounted in 50% glycerol in TBS, pH 8.1, and 6 mg/ml N -propyl gallate. For colocalization of nucleation sites and Arp3 or p21, cells were permeabilized in the presence of rhodamine-labeled actin and fixed with formaldehyde as above. The samples were treated further with methanol for 2 min before being processed for immunolabeling as described in the immunofluorescence protocol above, with the Triton permeabilization step omitted. Images were taken using constant settings on an Olympus IX70 microscope with 60× NA 1.4 infinity-corrected optics coupled to a computer-driven cooled CCD camera using IPLab Spectrum software (VayTek). The digitized images were converted linearly in NIH Image (program developed at the National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/ ) and analyzed using different macros. For measurement of the fluorescence from the leading edge back to ∼3 μm inside the cell, the macro gives the mean of pixel intensity within 1 pixel concentric perimeter, running from the outside of the cell to the inside . For the kinetic experiment, rhodamine fluorescence was expressed as the mean pixel intensity within a 1.1-μm band covering the whole cell perimeter at the leading edge. As shown previously , lamellipods are flat and of uniform thickness so that variations in cell thickness do not contribute to fluorescence signal intensity. The same results were obtained using conventional imaging, confocal or digital deconvolution methods. Biotin-labeled actin was prepared according to Okabe and Hirokawa with modifications. 25 mg of G-actin (alpha/rabbit skeletal muscle) was dialyzed for 24 h in depolymerization buffer (2 mM Tris-HCl, pH 7.5, 0.1 mM CaCl 2 , 0.2 mM ATP). It was clarified for 20 min at 95,000 rpm in a centrifuge (TL100 Ultracentrifuge; Beckman), diluted to 3 mg/ml in depolymerization buffer above, and polymerized at room temperature for 2 h by adding final concentrations of: 10 mM Tris-HCl, pH 7.5, 2 mM MgCl 2 , 100 mM KCl, and 1 mM ATP. 8 mg N -hydroxysuccinimidobiotin ( Pierce Chemical ) was added to the solution and biotinylation was allowed to proceed for 10 min at room temperature. The reaction was quenched by adding 100 mg of sodium glutamate and F-actin was pelleted. Afterwards, F-actin was run through two cycles of polymerization/depolymerization, where the G-actin suspension was polymerized by adding 2 mM MgCl 2 , 50 mM KCl, 10 mM Pipes, pH 7, in a water bath at room temperature for 1 h, and depolymerized in buffer A (2 mM Tris, pH 8, 0.2 mM CaCl 2 , 0.02% NaN 3 , 0.2 mM ATP, 0.5 mM DTT). Actin was purified on a gel filtration column (Sephadex G180; Pharmacia ) in buffer A. Storage was done in liquid nitrogen in 1 M sucrose. The initial coupling ratio was ∼2 mol (biotin)/mol (actin), but some loss occurred on storage. The biotin-labeled actin had a polymerization activity comparable to that of unlabeled actin, both in terms of percent polymerizable at equilibrium (>90%) and polymerization rates (as measured by viscometry). b-GA2 was prepared as described previously using G-actin labeled with biotin as described above. For immunoelectron microscopy and colocalization with the Arp2/3 complex, 100 nM of the complexes was used in place of biotin–actin in the permeabilization buffer and the preparations were further processed as described below. MTLn3 cells were grown on formvar or parlodion–carbon-coated gold square support grids (Electron Microscopy Science) on coverslips for 18–24 h. The immunoelectron microscopy was based on a previously described protocol with some modifications. Coverslips were treated with 0.25% Triton X-100 in buffer C in the presence of 1% BSA and 0.45 μM biotin-labeled actin for 1 min. After a rapid wash in buffer C, the preparations were fixed with 0.5% glutaraldehyde in cytoskeleton stabilization buffer, pH 6–6.1, in the presence of 5 μM phalloidin for 10 min. The grids were rinsed in cytoskeletal buffer and incubated for 15 min in 50 mM NH 4 Cl in PBS (145.5 mM NaCl, 4 mM NaH 2 PO 4 , 6 mM Na 2 HPO 4 · 7 H 2 O), with a change to fresh solution after 7 min. This was followed by a 30-min incubation in 0.1% gelatin (IGSS gelatin; Amersham ) in PBS, with a change to fresh solution after 15 min. The grids were incubated for 6 h with 5 nm gold-conjugated antibiotin antibodies. They were washed three times for 10 min in 0.1% gelatin, 1 min in 0.05% Triton in PBS, and four 1-min washes in PBS. They were postfixed in 1% glutaraldehyde/5 μM phalloidin in cytoskeleton stabilization buffer for 15 min, and transferred briefly in cytoskeleton stabilization buffer before being negatively stained. For double labeling the actin and Arp2/3 complex, grids were treated additionally with methanol for 2 min before the NH 4 Cl step, blocked for 30 min with gelatin, and incubated for 1.5–2 h with anti-Arp3 or anti-p21 antibodies. The grids were rinsed three times for 10 min in 0.1% gelatin and incubated for 6 h in a mixture of 5 nm gold-conjugated antibiotin antibodies and 10 nm gold-conjugated anti–rabbit antibodies. They were washed and postfixed as described above. For negative staining, the grids were transferred sequentially through four drops of 40 μg/ml bacitracin in water and four drops of 1% phosphotungstic acid. The grids were blotted dry and observed using 100× transmission electron microscope (JEOL USA, Inc.) at 80 kV. MTLn3 cells were grown on 5-mm glass coverslips and processed for immunoelectron microscopy as described above. For better preservation of the actin cytoskeleton, methanol treatment during the double labeling with anti-Arp2/3 complex antibodies was done with 70% methanol. After the postfixation step, coverslips were rinsed three times in water and processed for FDS based on the procedure described by Hartwig . Briefly, fixed coverslips were washed with two changes of distilled water. They were placed on a specimen mount of the rapid freezing apparatus (CF100; Life Cell Corp.) and frozen by slamming them into a liquid nitrogen–cooled copper block. Freezing tabs containing the frozen coverslips were transferred to a liquid nitrogen–cooled stage of a freeze fracture apparatus (CFE-50; Cressington), the stage temperature raised to −90°C for 90 min, and rotary shadowed at a 45° angle with 1.2–1.3 nm tantalum-tungsten, and 2.5 nm carbon at 90°. Replicas were separated from the coverslip with 25% hydrofluoric acid, washed into distilled water, and picked up on the surface of formvar-coated copper grids. The samples were observed using a JEOL 100CX transmission electron microscope at 100 kV. Images were viewed as negatives for better contrast, the gold particles appearing white. Morphometric analysis was done on randomly chosen intact cytoskeletons. Leading edges of lamellipodia were identified at low magnification and their typical lamellipodial character, as defined by the presence of a dense orthogonal network of actin filaments , was confirmed at high magnification. In some instances, two different areas of the cell periphery were analyzed for unstimulated cells: nonlamellipodial areas, representing cell edges that did not possess the characteristics of lamellipodia ; and typical leading edges . Unless those two categories are mentioned specifically, all data for unstimulated cells (EGF0) were obtained from typical lamellipodial areas. Quantitation was done using blind or double-blind methods. The negatives were scanned at high resolution and the digitized images were transferred to NIH image. A macro was written that enabled us to analyze the distribution of the gold particles: within a given area, the position of each particle was marked and we counted the particles in contiguous boxes. The size of the boxes was adjustable so that we could analyze the distribution of the particles along the leading edge (not shown), or across the leading edge. Typically boxes 2-μm wide were run at 0.1-μm steps into the leading edge, starting outside the cell . Two different sizes of particles could be analyzed simultaneously on the same image. This allowed direct colocalization of actin and Arp2/3 on the same image, with the same reference for the membrane. Since the cells were Triton-permeabilized and exogenous actin had polymerized onto preexisting filaments, the membrane position was assigned as the external edge of the dense filament network at the leading edge. This was done easily and reproducibly at low magnification . (A) Analysis of the distribution of the nucleation sites and/or Arp2/3 complex . Consecutive boxes of 2 × 0.1 μm were drawn at the leading edge from the outside to the inside of the cells and the number of particles in each box was plotted as a function of the distance of the box from the membrane. (B) Analysis of the filament density at the leading edge . Five lines were drawn perpendicular to the membrane at the leading edge and the filaments crossing the lines were marked. The macro described above was run with 0.1-μm steps along the lines and the total number of filaments crossing each of the five lines was counted in each 0.1 μm interval. (C) Analysis of filament length . Measurements were done using the standard length feature in NIH Image. Since most of the actin network at the leading edge is within a single plane (only a small proportion of the filaments are growing perpendicular to the lamellipod), the tracings and corresponding measurements were done in two dimensions only as shown in Fig. 7 G. Two complementary sets of data were generated: the global filament population terminating within 0.5–1 μm at the leading edge was analyzed, both filaments with a free end and filaments within the network with no free end (but showing distinctive origin and end at the intersection with another filament) were followed carefully inside the network to their origin. The second analysis involved exclusive measurements of filaments with one free end terminating within 0.2–0.3 μm at the leading edge and presenting an easily identifiable origin . The F-actin concentration at the leading edge was calculated from the total length of filaments within a 1 × 1 μm square at the leading edge, assuming a thickness of 176 ± 14 nm for the lamellipod in that particular zone (Lanni, F., personal communication), and a monomer size within the filament of 2.75 nm . MTLn3 cells stimulated with EGF undergo a broad lamellipod extension which is maximal within 3 min and driven by actin polymerization at the leading edge . Using this well characterized model, we investigated the relationship between sites of actin polymerization and Arp2/3 complex location in leading edges. We used antibodies raised against three different subunits of the Arp2/3 complex to analyze the distribution of the Arp2/3 complex in these cells. We first analyzed the localization of the Arp2/3 complex in MTLn3 cells in reference to actin distribution. Since these anti-Arp2/3 complex antibodies all require methanol fixation, which alters phalloidin binding, actin was visualized in the cells using anti-actin antibodies. The pattern obtained for actin using anti-actin antibodies was virtually identical to the F-actin pattern revealed by fluorescent phalloidin labeling (data not shown). Immunofluorescence data show that the Arp3 and p21 subunits of the Arp2/3 complex colocalize with actin at the leading edge of these stimulated cells . Although the signal was somewhat weaker, p34 showed a similar distribution (data not shown). None of the proteins localized to stress fibers, though occasional weak staining of stress fibers was noticed with the anti-Arp3 antibodies . Some cytoplasmic staining was also observed, including staining in discrete particles that also contain F-actin, as demonstrated by FITC-phalloidin staining (data not shown). The nuclear staining represented mostly nonspecific binding of the secondary antibodies. Quantitative analysis of the distribution of actin and Arp3 or p21 at the leading edge showed very similar localization, both protein concentrations maximizing within <1 μm at the leading edge . We demonstrated previously that EGF-stimulated lamellipod extension is dependent on actin polymerization at the leading edge . By permeabilizing stimulated cells in the presence of 0.45 μM rhodamine-labeled actin stimulation, the sites of actin nucleation (i.e., free barbed ends) can be visualized directly . Since 0.45 μM is below the critical concentration for the pointed end , only free barbed ends are visualized . As the polymerization of exogenous G-actin is not diffusion limited , exogenous and endogenous G-actin must be exchanged rapidly, and endogenous G-actin should fall to negligible levels quickly after permeabilization. This is consistent with the uniformity of rhodamine–actin incorporation into the leading edge observed in our experiments that would not occur if there were punctate pools of endogenous G-actin released during permeabilization. Under the current experimental conditions, most of the nucleation activity is localized within <2 μm at the leading edge after stimulation (see below). Quantitative analysis of fluorescence within that specific zone at the leading edge gives an accurate view of the kinetics of appearance of the nucleation sites: EGF stimulation generates a transient increase in nucleation activity, peaking sharply at 50 s, and generates an average 2.4-fold increase in nucleation activity at the leading edge . Nucleation activity returns to residual levels 5–6 min after stimulation. We performed direct colocalization of these active nucleation sites and the Arp3 subunit in MTLn3 cells after stimulation . In unstimulated cells (EGF0), Arp3 is enriched in the peripheral submembraneous compartment, in conjunction with nucleation activity, and in ruffling areas. After EGF stimulation (EGF1), Arp3 is recruited homogeneously to the extreme edge of cells in conjunction with newly created nucleation sites . After 3 min (EGF3), nucleation activity remains confined to the very submembraneous compartment and the tips of the stress fibers (presumably focal contacts). Arp3 distribution also is restricted mainly to the outermost part of the leading edge but sometimes tends to extend further inside the cell, beyond the nucleation site location. Some particulate staining is seen in these conditions that correspond to small dorsal protrusions containing F-actin, Arp3, and nucleation sites . These are similar to the Arp-, capping protein-, and actin-containing dorsal protrusions recently described on fibroblasts . The above results showed that Arp2/3 complex and nucleation sites colocalize at the leading edge, but the resolution of light microscopy (∼0.3 μm) was not sufficient to determine if they actually overlapped. To study the distribution of the nucleation sites at the leading edge of lamellipods at high resolution as well as their spatial and temporal relationship with the Arp2/3 complex, we adapted the protocol used to visualize nucleation sites at the light microscope level and used biotin-labeled actin to visualize nucleation sites at the electron microscope level. We studied the leading edge where the lamellipod is flat enough to allow unequivocal identification of actin-containing structures relative to the plasma membrane. To obtain a comprehensive view of the cytoskeleton's ultrastructure at the leading edge, independent of technique-specific artifacts, we used both negative staining and FDS techniques. The kinetics, amplitude, and localization of the nucleation activity obtained with these techniques were strikingly similar to those measured at the light microscope level, as well as highly reproducible (data not shown). We used only light permeabilization (low detergent concentration and short extraction time) to minimalize extraction. Under such conditions, even omitting the exogenous actin in the permeabilization step does not affect the structure of the cytoskeleton at the leading edge markedly . Negatively stained images show that the cytoskeleton at the leading edge is arranged as a dense network of filaments . In unstimulated cells, two types of cytoskeleton organization are found: cell edges in nonlamellipodial areas (not organized as leading edges) contain loose networks of long filaments with little, if any, exogenous biotin-labeled actin incorporation ; and typical leading edges of dense filament networks where individual filaments can be seen growing radially from the edge, with some exogenous biotin-labeled actin incorporation . After stimulation, a large proportion of the cell periphery is arranged as a typical leading edge with a broad peripheral lamellipod extension . The filaments form a denser network of actin where intense biotin-labeled actin incorporation has occurred as a result of the increase in nucleation activity . Morphometric analysis of the distribution of the nucleation sites was conducted using a macro running in NIH Image (see Materials and Methods). With the membrane position set as the extreme edge of the lamellipod when viewed at low magnification , the macro enabled us to count the gold particles in contiguous 2 × 0.1 μm 2 boxes from the outside to a few microns inside the cell . The resulting distribution was plotted . On the same negatives, the filament density at the leading edge was evaluated, with the same membrane reference as that taken for the gold particle counts . Quantitation of nucleation activity as biotin–actin density at the leading edge confirms a transient increase 1 min after stimulation and a rapid decrease back to resting levels after 3 min . The maximum nucleation activity generated after stimulation is confined within a 0.2–0.3 μm zone, directly at the membrane, and only residual (background) nucleation activity remains >1–1.5 μm away from the membrane . As opposed to the sharp location and transient generation of nucleation sites, EGF stimulation results in an ∼1.5-fold increase in filament density within a 1.5–2-μm zone adjacent to the membrane. This high density remains for 5 min after stimulation . Thus, the maximum nucleation activity generated after stimulation is confined to a narrower region than the one covered by the high filament density zone generated after stimulation. To get greater insight into the three-dimensional filament architecture at the leading edge, we used the FDS technique. This technique allows a very high resolution of the cytoskeleton with minimum reorganization of the actin filaments, potentially minimizing the generation of artifacts. The results obtained using this technique were entirely consistent with those acquired on negatively stained samples. Furthermore, the diameter of the filaments observed in the replicas was routinely <11 nm, indicating that a high degree of resolution was achieved with minimal metal deposition. We analyzed the filament architecture at the leading edge of the cells before and after stimulation with EGF. As with negative staining, leading edges could be identified easily by their typical orthogonal arrangement of filaments with a denser zone at the extreme edge , as opposed to loose bundles of parallel filaments in other parts of the cells . The 5 nm gold particle distribution reveals filaments that have incorporated biotin–actin, localizing the active nucleation sites at the extreme edge of the cells , as shown previously with negative staining . The network of actin filaments was denser after stimulation . Different types of filament crossing and/or branching were observed including: branching with an angle of ∼70°, typical of the in vitro branching observed for Arp2/3 complex on filaments growing radially at the leading edge or within the network; T-branching inside the network at the leading edge ; and Y-branching and filament branching with smaller angles . To evaluate the concentration of F-actin at the leading edge, the total length of actin filaments was evaluated in a 1 × 1 μm 2 box at the leading edge, and was found to be 66 ± 6 μm/μm 2 . Assuming 176 ± 14 nm for the thickness of the lamellipod at the leading edge (Lanni, F., personal communication), this gave us an approximate concentration of 9.3 mg/ml for F-actin at the leading edge. We measured the length of the filaments within 1 μm of the leading edge (see Materials and Methods). Two different sets of data were generated corresponding either to the total filament population at the leading edge , or a more restricted compartment corresponding exclusively to filaments with one free end . Both sets of data show an average 30% decrease in filament length at the leading edge 1 min after EGF stimulation compared with unstimulated cells (Table I , nonlamellipodial and lamellipodial areas combined). This is consistent with measurements made on negatively stained samples showing 32 and 28% decreases in length at 1 and 3 min, respectively (data not shown). The filament length distribution within a 1-μm zone behind the membrane of resting cells (nonlamellipodial-type and typical leading edges combined) is widely distributed with filaments ranging in size from 30 to 1,000 nm . In contrast, after EGF stimulation the cell periphery consists of broad lamellipodial structures with typical leading edges containing mainly short filaments, ranging from 30 to <300 nm . This particular arrangement of short filaments persists after 3 min of stimulation , and is consistent with the maintenance of a high filament density . The specific loss of long filaments that accompanies the general decrease in filament length after stimulation strongly suggests the intervention of a filament severing activity. The Arp2/3 complex is present at the leading edge and shows the distribution expected . It can be identified on the sides of filaments and at filament intersections . As a control, the distribution of another cross-linking protein, ABP280, was also analyzed. As expected from previous work , ABP280 is present at the leading edge where it localizes at filament intersections, and unlike Arp2/3 complex, does not show any obvious side binding (data not shown). Colocalization of the Arp2/3 complex (Arp3 and p21 subunits) and the nucleation sites was performed on negatively stained samples using the morphometric analysis described above. For each cell, Arp2/3 (10 nm gold particles) and the nucleation sites (5 nm gold particles) were measured on the same image with the same reference for the position of the membrane (determined as above). The amount of Arp3 at the leading edge approximately doubles 1 min after EGF stimulation and is distributed as a broad peak starting ∼100 nm back from the membrane . 3 min after stimulation, the amount of Arp3 at the leading edge is slightly reduced, but is still higher than before stimulation. p21 distribution follows the same trend as Arp3 . During this 3-min interval of stimulation with EGF, the membrane has moved ∼5.2 μm to the left (data not shown). To refine our positioning of the Arp2/3 complex relative to the barbed ends and obtain a precise count of the number of barbed ends, we used a different method to localize barbed ends. Since our usual method involves the growth of the actin filaments from exogenous actin, we designed an experiment where we could prevent actin polymerization, and label barbed ends directly where they are by capping them with b-GA2. As shown in Fig. 10 , under these conditions the edge of Arp3 distribution is still offset from the membrane, whereas the barbed end distribution remains highly localized at the membrane. In addition, the concentration of the Arp2/3 complex at the leading edge barely doubles after stimulation, whereas there is a fivefold increase in barbed ends. Although the extreme edge of MTLn3 cells has some features comparable to those of constitutively moving cells such as keratocytes , the general organization of the actin cytoskeleton in these carcinoma cells more closely resembles that of chemotactic ameboid cells such as macrophages , leukocytes , and Dictyostelium discoideum . In unstimulated cells, the cytoskeleton at the periphery is arranged as a loose network of long, occasionally bundled, filaments. Some cells are polarized and present a leading edge where filaments are arranged in a 1–1.5-μm wide, high density orthogonal network, similar to the leading edge of unstimulated fibroblasts . The density of filaments rapidly decreases a few microns away from the edge. After stimulation, the density and width of the orthogonal network at the edge increases and a clear zone, mostly devoid of actin filaments, is created behind the leading edge as the lamellipod advances. These are features that are not observed in keratocytes . The network at the leading edge of MTLn3 cell is composed of tightly entangled interwoven filaments, featuring multiple intersections of two or more filaments, and its complexity increases with filament density after stimulation. Most of the filaments inside the network are relatively short (0.2–0.3 μm on average before stimulation and even less after stimulation). This is slightly shorter than previously reported filament lengths measured on quick frozen deep-etched macrophages , or inferred from depolymerization kinetics in leukocytes . However, it is remarkably similar to what has been measured in stimulated platelets or in Dictyostelium , where the mean filament length was 0.2 μm with a large proportion of small (<140 nm) filaments . The Arp2/3 complex can generate 70° angle branches between filaments in vitro . In vivo, a large subset of the branches observed in the cytoskeleton of keratocytes displays similar 70° angles . This has drawn others to the conclusion that Arp2/3 could be a major component of cytoskeleton organization by nucleating and branching actin filaments . Although we have noticed some 70° branching at the leading edge of MTLn3 cells, the organization of the cytoskeleton that we observed is more typical of what has been described in chemotactic ameboid cells, with a more complex set of branching, including T-, Y-, and X-branching . This morphology does not appear to be technique dependent, but rather common to chemotactic cells as seen by different techniques such as critical point drying , FDS , or negative staining . Although we have localized the Arp2/3 complex at filament vertices, other cross-linkers like ABP280 are present at the leading edge (data not shown) where they can contribute to maintain the integrity of the cytoskeleton and define filament branching . Indeed, the three-dimensional architecture of the cytoskeleton, the distance between filament intersections (data not shown), and the actin concentration that we measured at the leading edge are consistent with a network of actin and filamin-type cross-linkers . The relative contributions of these different molecules to filament organization might differ from one type of cell to the other, which could explain why a 70° Arp2/3 branching pattern is more common in the keratocyte . Alternatively, Arp2/3 branching might be more involved in constitutive movement, as shown in keratocytes, as opposed to transient and rapid reorganization of the cytoskeleton after EGF stimulation. Interestingly, we also see the Arp2/3 complex along the sides of filaments in the absence of any obvious branching, in agreement with previous in vitro observations . EGF stimulation triggers a transient increase in nucleation activity that is highly controlled in terms of kinetics (sharp peak at 50–60 s) and localization (within 100–200 nm at the extreme edge of the lamellipod). The position of the nucleation sites remains constant with respect to the membrane of the extending edge as the cells form lamellipods. The increase in nucleation activity after stimulation is accompanied by an increase in filament density at the leading edge, covering a zone extending up to 2–2.5 μm further inside the cells. Both light and electron microscopy show that the Arp2/3 complex is localized within this network, where its concentration increases after EGF stimulation. Although a subset is present close to the membrane where the nucleation sites are, the distribution of the Arp2/3 complex is not restricted to the 100–200-nm polymerization zone at the membrane, but extends 1.5–2 μm inside the dense F-actin network both before and after stimulation. Furthermore, in most cases, the increase in Arp2/3 concentration at the membrane does not reflect the increase in nucleation activity: the Arp2/3 amount never increases more than twofold in any of our experiments, whereas up to fivefold increases were observed for the number of free barbed ends . 1 min after stimulation, the Arp2/3 concentration is maximum close to the membrane, and decreases back to the level measured in unstimulated cells within a distance of 2 μm from the growing edge. This corresponds to the site where the membrane was located before stimulation (since the lamellipod has extended 2.2 μm at 1 min and 5.2 μm at 3 min after stimulation, data not shown). Thus, we propose that the Arp2/3 distribution that we observed is the result of the progressive binding of Arp2/3 to filaments as the leading edge advances. The Arp2/3 complex is recruited to filaments under the membrane as the nucleation activity increases but, unlike the nucleation sites that continue to advance with the membrane, the majority of the Arp2/3 complex stays in place. This suggests that Arp2/3 is functioning as a pointed end capper, which would explain the broad peak of Arp2/3 distribution compared with the sharp peak of nucleation sites at the leading edge. The Arp2/3 complex caps the pointed ends of actin filaments with high affinity . Could the Arp2/3 complex have only a passive role of pointed-end capping at the leading edge? In this model, the increase in Arp2/3 concentration at the leading edge after stimulation simply could reflect the increase in pointed ends that we observed . However, the Arp2/3 distribution does not follow exactly the filament distribution, as it tends to increase more than the filament number does, and drops more rapidly than the filament density within 2 μm of the leading edge. While we cannot rule out a simple pointed-end capping role for Arp2/3, it seems likely that the complex has a more active role in actin polymerization. Altogether, the increase in the number of pointed ends observed after stimulation , the decrease in filament length, and disappearance of the long filaments shown here rule out uncapping as the dominant mechanism for generating the increase in nucleation activity observed at the leading edge after EGF stimulation. Nonetheless, uncapping could be the mechanism responsible for generating the nucleation sites that we see at the ends of the stress fibers, since the growth happens only at the extreme ends of the stress fibers (i.e., not consistent with severing) and does not seem to involve any Arp2/3 complex recruitment. The other two mechanisms capable of generating new nucleation sites after stimulation are de novo nucleation or severing (or both combined). Both mechanisms could account for the decrease in filament length that we observed: the former because it might generate a large population of filaments that will be shorter than the preexisting ones, and the latter because it actually cuts the preexisting filaments into smaller ones. However, the disappearance of the longer filaments that we observed immediately after stimulation is highly suggestive of a severing mechanism. Original work on the Arp2/3 complex suggested that it could nucleate actin polymerization at the surface of the bacteria, Listeria monocytogenes . However, the Arp2/3 complex probably is not a true nucleator, but rather captures and stabilizes unstable actin dimers . The rate constants of assembly/disassembly of actin monomers into a dimer are such that the probability of generating actin dimers from free monomers or monomers bound to sequestering proteins in vivo is essentially null. Similarly, the formation of a complex between Arp2/3 and one actin monomer, as well as addition of a second actin monomer to this complex to create a nucleus is extremely unlikely . However, capture and stabilization of actin dimers or oligomers by the capping of pointed ends by the Arp2/3 complex is a very efficient process that can lead to the stabilization of nucleation sites for actin polymerization. We propose that a severing activity occurs after stimulation that rapidly generates actin oligomers stabilized by the Arp2/3 . A likely candidate for this severing activity is cofilin, which can generate severed filaments with free barbed ends. Cofilin activity is regulated by pH as well as its phosphorylation status, the phosphorylated form being unable to bind actin. Chemotactic peptides and PMA in neutrophils, as well as growth factors in epithelial cells, induce a dephosphorylation of cofilin that is associated with a shift of cofilin into F-actin–rich areas at the cell periphery. This suggests that cofilin could be a major component in generating nucleation activity after stimulation . Recently, the kinase that turns off cofilin activity has been identified as LIM kinase-1. Moreover, some data suggest that LIMK-1 is a component of the small, GTP protein rac signal transduction pathway leading to lamellipodia formation in fibroblasts . We have evidence that cofilin is recruited at the edge of the lamellipodia in MTLn3 cells after EGF stimulation (Chan, A., personal communication) and that expression of a constitutively active LIMK-1 in those cells abolishes the EGF-stimulated increase in nucleation activity and subsequent lamellipod extension (Zebda, N., personal communication). Altogether, these results suggest that cofilin could have a major role in generating a transient increase in nucleation activity after EGF stimulation, by severing the filaments to create small actin oligomers with free barbed ends that would be stabilized by Arp2/3 complex to generate nucleation sites for actin polymerization . On the other hand, we cannot exclude the possibility that only a subset of the Arp2/3 complex at the membrane is transiently activated to nucleate actin filaments. This could explain why we observed a relatively low increase in Arp2/3 concentration at the leading edge after stimulation compared with the increase in nucleation activity. Indeed, it has been suggested that the Arp2/3 complex could be activated in vivo by different partners, which would greatly enhance its nucleating activity . Welch et al. have reported previously that the bacterial protein ActA could have such an effect. More recently, Machesky and Insall described two mammalian proteins, Scar1 and WASP, that interact with the Arp2/3 complex to regulate the actin cytoskeleton. Further work is required to decipher the sequence of molecular interactions that lead to actin polymerization and leading edge advance, and determine which mechanism allows the Arp2/3 complex to influence the outcome of these processes. | Study | biomedical | en | 0.999995 |
10209029 | MDCK cells were kindly provided by Dr. S. Tsukita (Kyoto University, Kyoto, Japan). NRK49F cells were purchased from American Type Culture Collection. pEF-BOS-HA-RhoA V14 , RhoA N19 , Rac1 V12 , Cdc42 V12 , pEF-BOS-myc-Rho-kinase , Rho-binding domain , RB/PH (TT) , and CAT (the catalytic domain of Rho-kinase, 6–553 aa) were constructed as described . pcDSRα-PKCβ lacking C1 domain at positions 6–159 aa was constructed as described . pQE-rat γ-adducin fragment (319–671 aa; 35H fragment-4D) was kindly provided by Dr. S. Jaken (University of Vermont, Burlington, VT), and 6 X His-tagged γ-adducin fragment was produced and purified as described . The expression plasmid of C3 transferase (pGEX-C3) was kindly provided by Dr. A. Hall (University College London, London, UK), and C3 was produced and purified as described . Glutathione- S -transferase (GST)-RB was produced and purified from Escherichia coli as described . GST-CAT was produced and purified from Sf9 cells as described previously . Rac1 N17 was produced and purified from E . coli as described . Anti-hemagglutinin (HA) monoclonal Ab (12CA5) was purchased from Boehringer Mannheim . [γ- 32 P]ATP was purchased from Amersham Corp. All materials used in the nucleic acid study were purchased from Takara Shuzo Corp. Other materials and chemicals were obtained from commercial sources. The cDNA encoding human α-adducin was inserted into the KpnI site of pEF-BOS-HA, into the KpnI site of pAcYM1-HA to obtain recombinant α-adducin by the use of a baculovirus system, and into the BamHI site of pGEX-2T. The cDNA encoding human β-adducin was also inserted into the KpnI site of pAcYM1-HA. The cDNAs of α-adducin T445A,T480A (AA) and α-adducin T445D,T480D (DD), which were substituted for Thr at both 445 and 480 amino acid residues by Ala or Asp, were generated with the use of a site-directed mutagenesis kit (Stratagene), and subcloned into pEF-BOS-HA, pAcYM1-HA, and pGEX-2T. The cDNAs of α-adducin T445A and α-adducin T480A were also generated as above and subcloned into pGEX-2T. RB/PH (TT) was expressed as a maltose-binding protein (MBP) fusion protein in E . coli and purified. GST-α-adducin, GST-α-adducin-AA, GST-α-adducin T445A , and GST-α-adducin T480A were produced and purified from E . coli . HA-α-adducin, HA-β-adducin, HA-α-adducin-AA, and HA-α-adducin-DD were produced in Sf9 cells. The cells expressing HA-α-adducin were suspended in homogenizing buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 10 μM A-PMSF, 10 μg/ml leupeptin). The suspension was sonicated and centrifuged at 100,000 g for 1 h at 4°C. The supernatant was applied onto a Mono Q column ( Pharmacia LKB Biotechnology ) which had been equilibrated with buffer A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT). The column was then washed, and the proteins were eluted with a linear concentration gradient of NaCl (0–600 mM) in buffer A. HA-α-adducin was eluted with ∼200 mM NaCl and purified to near homogeneity. HA-β-adducin, HA-α-adducin-AA, and HA-α-adducin-DD were prepared as HA-α-adducin. HA-α-adducin (70 μg of protein) was phosphorylated with GST-CAT (120 ng of protein) in 2 ml of kinase buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing 100 μM [γ- 32 P]ATP for 1 h at 30°C, and the reaction product was digested with Achromobacter protease-1 (AP-1) at 37°C for 20 h. The obtained peptides were applied onto a C18 reverse-phase column (4.6 × 250 mm; Shiseido, Japan) and eluted with a linear gradient of 0–48% acetonitrile for 100 min at a flow rate of 1.0 ml/min by HPLC (System Gold, Beckman). The radioactive peaks were separated and phosphoamino acid sequencing was carried out with a peptide sequencer (PPSQ-10; Shimadzu, Japan) as described . The fractions obtained from each Edoman degradation cycle were measured for 32 P in a Beckman liquid scintillation counter. The phosphopeptide Cys-Gln 440 -Gln-Arg-Glu-Lys-phospho-Thr-Arg-Trp- Leu-Asn-Ser 450 was chemically synthesized as an antigen by Peptide Institute Inc. Rabbit polyclonal antibody (anti-pT445) was raised against the phosphopeptide and affinity purified as described . To examine the specificity of anti-pT445, equal amounts of HA-α-adducin (40 fmol) with various ratios between phosphorylated and nonphosphorylated proteins were subjected to SDS-PAGE. HA-α-adducin-AA (40 fmol) was incubated in kinase buffer containing GST-CAT and ATP, and subjected to SDS-PAGE. The amount of phosphates incorporated into HA-α-adducin was simultaneously determined by [γ- 32 P]ATP. Immunoblot analysis was then performed with anti-pT445, anti-pT445 which was preincubated with a 100-fold amount of antigen-phosphopeptide, and anti-HA Ab. For some experiments, HA-α-adducin, HA-β-adducin, or 6 X His-γ-adducin fragment was separately phosphorylated with GST-CAT, and 40 fmol (as 32 P-incorporated amount) of phosphorylated proteins was subjected to immunoblot analysis with anti-pT445. MDCK cells and NIH3T3 cells were maintained in DMEM containing 10% calf serum, streptomycin, and penicillin. COS7 cells were maintained in DMEM containing 10% FBS, streptomycin, and penicillin. NRK49F cells were maintained in DMEM containing 5% calf serum and 1% nonessential amino acids. To obtain MDCK cells stably expressing HA-α-adducin, MDCK cells were transfected with pEF-BOS-HA-α-adducin along with pSVIISRα vector containing the neomycin resistance gene using Lipofectamine ( GIBCO BRL ) as described , and neomycin-resistant clones were selected. The transfection of plasmids into COS7 cells was carried out by the standard DEAE-dextran method. COS7 cells were cotransfected with pEF-BOS-HA-α-adducin and pEF-BOS-vector, pEF-BOS encoding HA-Rac1 V12 , HA-Cdc42 V12 , HA-RhoA V14 , HA-RhoA N19 , myc-Rho-kinase (full-length), HA-RhoA V14 and myc-Rho-kinase (full-length), HA-RhoA V14 and myc-RB, HA-RhoA V14 and myc-RB/PH (TT), myc-CAT, or pcDSRα-PKCβ. Separately, COS7 cells were cotransfected with pEF-BOS-HA-α-adducin-AA and myc-CAT. After a 24-h incubation, the transfected cells were incubated in serum-free medium for 24 h. The cells were treated with 10% (wt/vol) trichloroacetic acid. The resulting precipitates were subjected to immunoblot analysis using anti-HA Ab and anti-pT445. MDCK cells stably expressing HA-α-adducin were incubated in the absence or the presence of tetradecanoylphorbol-13-acetate (TPA) for 15 min followed by incubation in the presence of 0.1 μM calyculin A for 10 min. The cells were then treated as COS7 cells and subjected to immunoblot analysis. MDCK cells were deprived of serum for 24 h and incubated in DMEM containing 200 nM TPA or 50 pM recombinant human hepatocyte growth factor (HGF; Calbiochem ) for 15 min at 37°C. For cell scattering, the cells were not deprived of serum and incubated in DMEM containing 5% calf serum and 200 nM TPA for 2 h. The cells were fixed with 3.0% formaldehyde in PBS for 10 min and treated with PBS containing 0.2% Triton X-100 for 10 min. Phosphorylated adducin and F-actin were doubly stained with anti-pT445 and TRITC-phalloidin. For anti-pT445, the cells were then labeled with FITC-conjugated anti–rabbit Ig Ab. For a double immunofluorescence study, the clone stably expressing HA-α-adducin was stimulated as described above. Phosphorylated adducin and HA-α-adducin were doubly stained with anti-pT445 and anti-HA Ab. The cells were then labeled with FITC-conjugated anti–rabbit Ig Ab and Texas red–conjugated anti–mouse Ig Ab. The cells were examined using a Zeiss Axiophot microscope or a confocal microscope ( Carl Zeiss ). MDCK cells were seeded at a density of 2.5 × 10 3 cells per 13-mm cover glass in 60-mm tissue culture dishes and incubated for 8 h. Then, the cells were deprived of serum for 24 h. For microinjection, proteins were concentrated and during the concentration the buffer was replaced by microinjection buffer (20 mM Tris-HCl, pH 7.4, 20 mM NaCl, 1 mM MgCl 2 , 0.1 mM EDTA, 5 mM 2-mercaptoethanol). The indicated proteins were microinjected along with a marker protein (rabbit or mouse IgG at 0.5 mg/ml) into the cytoplasm of cells. Using microcapillaries (tip diameter is 0.5 μm), ∼3 × 10 −14 liter of each protein was microinjected by one injection. When C3 was microinjected at 0.1 mg/ml, the injected amount per cell was ∼3 × 10 −15 g (the intracellular concentration was estimated to be ∼0.15 μM). When HA-α-adducin mutants were microinjected at 5.0 mg/ml, the injected amount per cell was ∼150 × 10 −15 g (the intracellular concentration was estimated to be ∼2.0 μM, and it seemed to be much higher than the level of endogenous α-adducin). 30 min after the microinjection, the cells were incubated in DMEM containing 200 nM TPA for 15 min at 37°C. The cells were then fixed and stained with anti-pT445 or TRITC-phalloidin as described above. Cell migration assay using a wound healing model was performed as described previously . The confluent monolayer of the cells was scraped with a white chip. Soon after wounding, the medium was changed and indicated recombinant proteins were microinjected along with a marker protein (rabbit IgG, 1.0 mg/ml) into the cytoplasm of only the cells along the wound edge as described above. The original wound edge was determined by taking photographs soon after the cells were wounded and microinjected. After 6 h, the cells were then stained with TRITC-phalloidin, and the cells that moved (>20 μm) from the original wound edge were counted by taking photographs of the same fields as the original photograph. SDS-PAGE was performed as described . Protein concentrations were determined with BSA as the reference protein as described . We first determined the sites of phosphorylation of human α-adducin by Rho-kinase as follows. HA-tagged α-adducin, which was phosphorylated by Rho-kinase in the presence of [γ- 32 P]ATP, was digested with AP-I and subjected to reverse-phase HPLC as described . Two major radioactive peaks (named peak 1 and 2, respectively) were obtained and their amino acid sequences were determined. The sequence obtained from peak 1 was TRWLNSGRGDEASEEGQNGSSPK, corresponding to residues 445–467 of human α-adducin, and Thr445 turned out to be the phosphorylation site for Rho-kinase . The sequence obtained from peak 2 was EDGHRTSTSAVPNLFVPLNTNPK, corresponding to residues 475–497 of human α-adducin, and Thr480 turned out to be the phosphorylation site for Rho-kinase . Both phosphorylation sites were in the neck domain of α-adducin, and were different from previously identified sites of phosphorylation by PKA (Ser408, Ser436, Ser481, and Ser726) and by PKC (Ser726) . When GST-α-adducin T445A or GST-α-adducin T480A (substitution of Thr445 or Thr480 by Ala) was phosphorylated by Rho-kinase, GST-α-adducin T480A was more efficiently phosphorylated by Rho-kinase than was GST-α-adducin T445A . This result is apparently inconsistent with the observation that the radioactivity of peak 1 containing Thr445 is lower than that of peak 2 containing Thr480 in the HPLC analysis . This may be due to the different recovery of each peptide during separation by HPLC. Taken together, these results suggest that Thr445 is a preferential site of phosphorylation by Rho-kinase in vitro. An antibody that recognizes the phosphorylated state of a substrate at a specific site is a useful tool with which to evaluate site-specific phosphorylation in vivo and to visualize the cellular distribution of the protein phosphorylated at a specific site . We prepared and affinity purified the polyclonal antibody that recognized the Thr445-phosphorylated α-adducin (anti-pT445), against the synthetic phosphopeptide Cys-Gln-Gln-Arg-Glu-Lys-phospho-Thr 445 -Arg-Trp-Leu-Asn-Ser. An immunoblot analysis revealed that anti-pT445 specifically bound to HA-α-adducin phosphorylated by Rho-kinase in a dose-dependent manner, but not to nonphosphorylated HA-α-adducin or HA-α-adducin T445A,T480A (substitution of Thr445 and Thr480 by Ala; HA-α-adducin-AA) incubated with Rho-kinase and ATP . HA-α-adducin, which was phosphorylated by PKC or PKN at levels similar to that by Rho-kinase, was not recognized by anti-pT445 (data not shown). The binding of anti-pT445 to α-adducin phosphorylated by Rho-kinase was neutralized by preincubation of the antibody with the antigen phosphopeptide. Rho-kinase phosphorylated β- and γ-adducin in vitro as well . Neither β- nor γ-adducin phosphorylated by Rho-kinase was recognized by anti-pT445 . These results indicate that anti-pT445 specifically recognizes only α-adducin phosphorylated at Thr445. We examined whether α-adducin was phosphorylated via the Rho/Rho-kinase pathway in COS7 cells by immunoblot analysis with anti-pT445. cDNA of HA-α-adducin was cotransfected with plasmids carrying the cDNA of dominant active Rho family members, or dominant active or negative Rho-kinase . HA-α-adducin was almost equally expressed in all the HA-α-adducin–transfected cells. α-Adducin phosphorylated at Thr445 was undetectable in serum-starved COS7 cells expressing HA-α-adducin alone. The expression of dominant active RhoA (RhoA V14 ), a RhoA mutant that is defective in GTPase activity and thought to be constitutively the GTP-bound form in the cells, induced a small increment of the α-adducin phosphorylation at Thr445, whereas the expression of dominant negative RhoA (RhoA N19 ), a RhoA mutant that preferentially binds GDP rather than GTP and is thought to be constitutively the GDP-bound form in the cells, had minimal effects. Although the exact reason for the minimal increase in phosphorylated α-adducin in COS7 cells expressing RhoA N19 is unknown, it is likely that the overexpression of GDP-bound RhoA N19 induced the phosphorylation of α-adducin to a minimal extent because GDP · Rho also activates Rho-kinase in vitro to a small extent as described . Dominant active Rac1 (Rac1 V12 ) or dominant active Cdc42 (Cdc42 V12 ) had no effects. Under the conditions, RhoA V14 , RhoA N19 , Rac1 V12 , and Cdc42 V12 were almost equally expressed (data not shown). The expression of Rho-kinase induced the α-adducin phosphorylation to some extent. By the coexpression of RhoA V14 and Rho-kinase, the α-adducin phosphorylation was further enhanced. Rho-kinase is composed of catalytic (CAT), coiled-coil (COIL), Rho-binding (RB), and pleckstrin-homology (PH) domains . RB binds to Rho and inhibits the Rho-dependent Rho-kinase activity in vitro and in vivo . RB/PH (TT) , which is the carboxy-terminal portion of Rho-kinase and encompasses the RB and PH domains, has point mutations in the RB domain and does not bind to Rho . RB/PH (TT) inhibits the lysophosphatidic acid (LPA)-induced stress fiber formation in fibroblasts and neurite retraction in neuroblastoma cells , suggesting that RB/PH (TT) serves as the dominant negative form of Rho-kinase. Recently, we found that RB/ PH (TT) interacted with the catalytic domain of Rho-kinase and thereby inhibited the Rho-kinase activity in vitro without titrating out Rho (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, manuscript submitted for publication). We further found that RB/PH (TT) had no effect on catalytic activity of PKN or myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which is identified as a target of Cdc42 and is homologous to Rho-kinase within the kinase domain , in vitro (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, manuscript submitted for publication). RB/PH (TT) inhibited the Rho-kinase–induced stress fiber, but not MRCK-induced stress fiber in fibroblasts (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, manuscript submitted for publication). These observations indicate that RB/PH (TT) more specifically inhibits the Rho-kinase activity in intact cells. α-Adducin phosphorylation at Thr445 by RhoA V14 was suppressed by RB and RB/PH (TT) . Constitutively active Rho-kinase (CAT) greatly induced the α-adducin phosphorylation , whereas constitutively active MRCK did not (data not shown). When the dominant active form of PKC, which activates the TPA-responsive element linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester , was expressed, the phosphorylation of α-adducin was not detected by anti-pT445 . HA-α-adducin-AA coexpressed with CAT was not recognized by anti-pT445 . These results indicate that α-adducin was specifically phosphorylated at Thr445 via the Rho/ Rho-kinase pathway in intact cells. Both Rho and Rac are thought to be necessary for the HGF- and TPA-induced membrane ruffling and HGF-induced cell motility in MDCK epithelial cells, although neither RhoA V14 nor Rac1 V12 by itself induces membrane ruffling or cell motility . We examined the distribution of Thr445-phosphorylated α-adducin in the TPA- or HGF-stimulated MDCK cells. We have found by immunoblotting with isoform-specific antibodies that 120-kD α-adducin and 90-kD γ-adducin (alternatively spliced form of 80-kD γ-adducin) were expressed almost equally in MDCK cells. We have also found that 110-kD β-adducin was expressed to a lesser extent in MDCK cells. Therefore, it is likely that anti-pT445 recognizes only phospho-Thr445 in α-adducin in MDCK cells. The addition of TPA induced membrane ruffling at the outer edge of cell colonies as described . The immunoreactivity of anti-pT445 accumulated in the membrane ruffling areas, where F-actin and spectrin (data not shown) also accumulated. Phosphorylated α-adducin was also detected in the perinuclear region. Similar results were obtained when the cells were stimulated with HGF instead of TPA . The immunoreactivity with anti-pT445 was abolished by preincubation of the antibody with the antigen phosphopeptide (data not shown). In the nonstimulated MDCK cells, phosphorylated α-adducin was diffusely present in the cytoplasm, but not in the free ends of the plasma membrane. Between 2 and 16 h after the addition of TPA, the cells dissociated from each other and scattered, with polarized morphology and membrane ruffling in the leading edge as described . Phosphorylated α-adducin also accumulated in the leading edge of TPA-induced scattering cells . To compare the localization of phosphorylated α-adducin and total α-adducin, a double-label immunofluorescence study was performed in MDCK cells stably expressing HA-α-adducin. Immunoreactivity showing the distribution of HA-α-adducin (red) was detected strongly in the cell–cell contact sites , weakly in the membrane ruffling areas , and diffusely in the cytoplasm, as described . Phosphorylated α-adducin (green) was enriched in the membrane ruffling areas in the TPA-stimulated MDCK cells . The merged image of red and green immunofluorescence revealed the enrichment of phosphorylated α-adducin in the membrane ruffling areas and a small increment of phosphorylated α-adducin in the cytoplasm in the TPA-stimulated cells . Phosphorylated α-adducin did not accumulate at the cell–cell contact sites . Similar results were obtained when the cells were stimulated with HGF. To confirm that α-adducin is specifically phosphorylated at Thr445 during the action of TPA, immunoblot analysis with anti-pT445 was performed. The addition of TPA to the MDCK cells stably expressing HA-α-adducin increased the phosphorylation level of HA-α-adducin at Thr445 . We next examined whether the Rho/Rho-kinase pathway was required for the TPA-induced membrane ruffling. The microinjection of C3, which is thought to interfere with endogenous Rho functions, RB, or RB/PH (TT) inhibited the TPA-induced membrane ruffling and the accumulation of phosphorylated adducin in the free end of the plasma membrane ; membrane ruffling was induced in 64% of TPA-stimulated cells in the outer edges of cell colonies, but in only 14% of cells injected with C3, 15% of cells injected with RB, and 22% of cells injected with RB/PH (TT). Dominant negative Rac (Rac1 N17 ) also inhibited the TPA-induced membrane ruffling to a similar extent . These results indicate that the Rho-kinase function is required for the TPA-induced membrane ruffling in MDCK cells. To determine whether the phosphorylation of adducin by Rho-kinase is necessary or sufficient for the membrane ruffling, we produced and used cDNAs encoding α-adducin mutants; HA-α-adducin T445A,T480A (HA-α-adducin-AA, substitution of Thr residues by Ala), which was not phosphorylated by Rho-kinase and expected to serve as the dominant negative form, and HA-α-adducin T445D,T480D (HA-α-adducin-DD, substitution of Thr residues by Asp), which was expected to mimic phosphorylated α-adducin and to serve as the constitutively active form . The microinjection of HA-α-adducin, HA-α-adducin-AA, or HA-α-adducin-DD by itself did not induce the membrane ruffling in MDCK cells (data not shown). This observation is consistent with the notion that RhoA V14 alone does not induce the membrane ruffling, and that a lot of processes are required for the induction of membrane ruffling. HA-α-adducin or HA-α-adducin-DD was translocated to the membrane ruffling area (data not shown) and did not inhibit the TPA-induced membrane ruffling, whereas HA-α-adducin-AA inhibited the ruffling; membrane ruffling was induced in ∼60% of TPA-stimulated cells injected with HA-α-adducin or HA-α-adducin-DD, but in only 24% of cells injected with HA-α-adducin-AA . Although the inhibitory mechanism of HA-α-adducin-AA is not known, HA-α-adducin-AA may substitute for endogenous α-adducin to oligomerize with endogenous β- or γ-adducin, and thereby stay as an inactive complex to inhibit functions of endogenous adducins. Essentially similar results as to the effects of RB/PH (TT) or HA-adducin-AA on membrane ruffling were obtained when MDCK cells were stimulated with HGF instead of TPA (data not shown). To confirm that HA-α-adducin-AA specifically inhibits the function of endogenous α-adducin but not those of other substrates of Rho-kinase such as MLC or the ERM family proteins, we examined whether the inhibitory effect of HA-α-adducin-AA on the membrane ruffling in MDCK cells was reversed by coinjection of HA-α-adducin or HA-α-adducin-DD. As shown in Fig. 5 C, HA-α-adducin reversed the inhibitory effect of HA-α-adducin-AA in a dose-dependent manner. HA-α-adducin-DD more efficiently reversed the inhibitory effect. These results indicate that HA-α-adducin-AA specifically inhibits the function of endogenous α-adducin but not those of other substrates. When HA-α-adducin-AA was microinjected into NIH3T3 cells, HA-α-adducin-AA did not affect the LPA-induced formation of stress fibers, which is mediated by MLC phosphorylation through the Rho/Rho-kinase pathway , whereas RB/PH (TT) inhibited the LPA-induced formation of stress fibers as described . HA-α-adducin-AA did not affect the LPA-induced formation of microvilli, which is mediated by ERM phosphorylation through the Rho/Rho-kinase pathway, under the conditions in which RB/PH (TT) inhibited the LPA-induced formation of microvilli . Taken together, these results suggest that HA-α-adducin-AA specifically interferes with the pathway mediated through the α-adducin phosphorylated by Rho-kinase in intact cells. When HA-α-adducin-DD, but not HA-α-adducin-AA, was comicroinjected with RB or RB/PH (TT), the inhibitory effect of RB or RB/PH (TT) on the TPA-induced membrane ruffling was counteracted . Under these conditions, HA-α-adducin also counteracted the inhibitory effect of RB/PH (TT), but not that of RB . This difference is likely due to the distinct mode of action between RB and RB/PH (TT). RB/PH (TT) interacts with the catalytic domain of Rho-kinase and inhibits the activity of Rho-kinase in a manner competitive with peptide substrates of Rho-kinase such as RRRLSSLRA without titrating out Rho (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, manuscript submitted for publication). The excess of HA-α-adducin over RB/PH (TT) in the cells may neutralize a competitive inhibition of Rho-kinase by RB/PH (TT), and then HA-α-adducin can be phosphorylated by the activated Rho-kinase. Consistently, when RB/PH (TT) was microinjected at higher concentrations, HA-α-adducin could not counteract the inhibitory effect of RB/PH (TT), whereas HA-α-adducin-DD was still capable of counteracting (data not shown). In contrast to RB/PH (TT), RB binds to the effector domain of Rho, and is expected to inhibit the interaction of Rho-kinase with Rho and thereby the activation of Rho-kinase by Rho . Thus, HA-α-adducin may not overcome the inhibitory effect of RB in MDCK cells. On the other hand, because HA-α-adducin-DD can mimic α-adducin phosphorylated at Thr445 and Thr480 in vivo, HA-α-adducin-DD may bypass Rho-kinase to induce membrane ruffling during the action of TPA irrespective of the Rho-kinase activity. HA-α-adducin-DD counteracted neither the inhibitory effect of C3 nor that of Rac1 N17 . GTPγS-bound RhoA I41 , which is insensitive to C3, could completely overcome the inhibitory effect of C3 as described . Taken together, these results indicate that HA-α-adducin-DD serves as the constitutively active form, and that α-adducin is a crucial substrate of Rho-kinase downstream of Rho for membrane ruffling. Membrane ruffling is thought to be an essential event in cell motility . We then investigated whether the α-adducin phosphorylation by Rho-kinase is involved in cell motility. C3 has been shown to inhibit the wound-induced migration of IEC-6 cells . We performed a wound healing assay by the use of NRK49F cells to investigate roles of Rho-kinase and adducin in cell motility, since it was difficult to estimate the precise distance moved in MDCK cells and to evaluate cell motility induced by TPA or HGF, and NRK49F cells were more suitable for a wound healing assay than were MDCK cells. Linear wounds were made in a confluent monolayer of NRK49F cells and ∼200 cells along the wound edge were microinjected with C3, RB/PH (TT), or the α-adducin mutants. 6 h after wounding, the cells that moved (>20 μm) from the original wound edge were counted. We also evaluated the wound-induced migration by a time-lapse recording. When MBP was injected as a control protein, 63% of the total injected cells showed wound-oriented migration, and ∼60% of the total injected cells had membrane rufflings . About 90% of the migrating injected cells had membrane rufflings in their leading edges . We found that α-adducin phosphorylated at Thr445 accumulated at the membrane ruffling areas in the leading edge and the perinuclear region in migrating NRK49F cells , as shown in MDCK cells. C3 and RB/PH (TT) inhibited the migration to the wound; only 24% of cells injected with C3 and 17% of cells injected with RB/PH (TT) migrated to the wound . The microinjection of HA-α-adducin-AA also inhibited the migration; only 21% of cells injected with HA-α-adducin-AA migrated to the wound . When C3, RB/PH (TT), or α-adducin-AA was injected, the percentages of the cells with membrane ruffling in the total injected cells (∼20%) were roughly the same as those of the migrating cells in the total injected cells (17–24%), and the stationary injected cells had few membrane rufflings . Whatever proteins were injected, the majority (∼90%) of migrating injected cells had membrane ruffling in the leading edge. These results suggest that the inhibition of cell migration by HA-α-adducin-AA, C3, or RB/PH (TT) correlates with the inhibition of membrane ruffling. In the HA-α-adducin– injected cells, the wound-oriented migration and membrane ruffling were not inhibited . HA-α-adducin-DD also inhibited the migration; 32% of cells injected with HA-α-adducin-DD migrated to the wound . However, the percentage of the cells with membrane ruffling in the total injected cells (∼60%) was much bigger than that of the migrating cells in the total injected cells (32%). The majority of migrating cells injected with α-adducin-DD had membrane rufflings in the leading edge, and about one-half of the stationary cells injected with α-adducin-DD had membrane rufflings . These results indicate that α-adducin-DD did not inhibit the membrane ruffling but did inhibit cell migration, suggesting that cycling between the phosphorylated and dephosphorylated states of α-adducin appears to be necessary for cell migration, but not for membrane ruffling. Consistently, RhoA V14 or CAT as well as Rac1 N17 inhibited the wound-induced migration (data not shown). In this study, we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480 in the neck domain. α-Adducin is phosphorylated at Ser408, Ser436, and Ser481 in the neck domain by PKA, and at Ser726 in the MARCKS-related domain by PKC and PKA . Among the in vitro phosphorylation sites of α-adducin, Thr445 and Thr480 are unique for Rho-kinase. We raised and used a site- and phosphorylation state–specific antibody for α-adducin. Anti-pT445 recognized HA-α-adducin phosphorylated by Rho-kinase, but not by PKC or PKN. Anti-pT445 recognized neither nonphosphorylated HA-α-adducin nor HA-α-adducin T445A,T480A incubated with Rho-kinase and ATP . These results indicate that anti-pT445 is a useful tool with which to detect the specific phosphorylation of α-adducin by Rho-kinase both in vitro and in vivo, distinguishing from the phosphorylation of α-adducin by PKC. Although Rho-kinase phosphorylated β- and γ-adducin in vitro , the phosphorylation sites have not been determined. Since the surrounding amino acid sequences of Thr437 in rat γ-adducin (QQREK T RWLNS) are completely identical to those of Thr445 in α-adducin (selected as antigen phosphopeptide for anti-pT445), the result that anti-pT445 did not cross-react with rat γ-adducin phosphorylated by Rho-kinase revealed that Rho-kinase does not primarily phosphorylate γ-adducin at Thr437. The surrounding amino acid sequences of Thr431 in β-adducin (QQKEK T RWLNT) are a little bit different from those of Thr445 in α-adducin. At this stage, we do not know whether Rho-kinase phosphorylates β-adducin at Thr431. Adducin is thought to be comprised of α- and β-, or α- and γ-adducin in vivo . We found that in MDCK and NRK49F cells a heterodimer of adducin which consists of α- and γ-adducin was mainly expressed (data not shown). Therefore, it is likely that anti-pT445 recognizes only phospho-Thr445 in α-adducin but not in β- and γ-adducin in both cells (see below). Further analysis is necessary to understand how the phosphorylation of β- or γ-adducin is regulated through Rho-kinase in vitro and in vivo. Using anti-pT445, we found that Thr445 phosphorylation of α-adducin occurred in a Rho/Rho-kinase-dependent manner in COS7 cells . We further found that α-adducin was phosphorylated at Thr445 in the TPA-stimulated MDCK cells . TPA also drives the activation of PKC. Indeed, TPA induces the phosphorylation of α-adducin at Ser726 in renal proximal tubule epithelial cells and MDCK cells . However, it is unlikely that PKC directly phosphorylates α-adducin at Thr445 in the TPA-stimulated cells, because anti-pT445 did not recognize α-adducin which was phosphorylated by PKC in vitro and was coexpressed with the dominant active PKC in COS7 cells. Rac and Cdc42 are also involved in TPA-induced phenomena such as membrane ruffling . It is possible that the protein kinases activated by Rac or Cdc42 phosphorylate α-adducin. However, we found that neither Rac1 V12 nor Cdc42 V12 induced the Thr445 phosphorylation of α-adducin in COS7 cells, and that the dominant active MRCK, which is identified as the target of Cdc42 and Rac1 (Nakamura, N., M. Amano, Y. Fukata, N. Oshiro, T. Leung, L. Lim, and K. Kaibuchi, manuscript in preparation), induced the phosphorylation of MLC but not of α-adducin at Thr445. Taken together, these results indicate that in intact cells α-adducin was specifically phosphorylated at Thr445 via the Rho/Rho-kinase pathway during the action of TPA. Although an in vitro experiment using GST-α-adducin T445A and GST-α-adducin T480A suggests that Thr445 is a preferential site of phosphorylation by Rho-kinase in vitro, at this stage it is unknown which of Thr445 or Thr480 is a preferential site in intact cells. Membrane ruffling, which is induced by several growth factors and is observed in the leading edges of motile cells, is thought to play an essential role in cell motility. The regulatory mechanism of the membrane ruffling is less well characterized. Not only Rac but also Rho is thought to regulate the membrane ruffling downstream of extracellular signals in certain types of cells, because C3 inhibits the TPA- or HGF-induced membrane ruffling in epithelial cells such as MDCK and KB cells . Here, we found that Thr445-phosphorylated α-adducin accumulated at the TPA-induced membrane ruffling areas and the leading edges of migrating cells . We furthermore found that RB and RB/PH (TT) as well as C3 inhibited the TPA-induced membrane ruffling, that α-adducin-AA also inhibited the membrane ruffling as the dominant negative α-adducin , and that α-adducin-DD specifically counteracted the inhibitory effect of RB and RB/PH (TT) on the membrane ruffling . These results indicate that α-adducin is a crucial substrate of Rho-kinase downstream of Rho for membrane ruffling. The fact that α-adducin-DD counteracted the inhibitory effect of RB but not that of C3 is probably due to difference between the inhibitory mechanisms by RB and C3. RB is the fragment of Rho-kinase which consists of the RB domain of Rho-kinase. RB binds to the effector domain of Rho, and then inhibits the interaction of Rho with Rho-kinase . Recent studies revealed that the various effector mutants of RhoA V14 , in which interaction with some effectors is maintained but with others is lost, make it possible to dissect the diverged pathways downstream of Rho. For example, RhoA Y42C abolishes interaction with PKN but maintains interaction with ROCK-I (an isoform of Rho-kinase) and RhoA F39L,C20R maintains interaction with PKN but abolishes interaction with ROCK-I . RhoA Y42C induces stress fiber formation, whereas RhoA F39L,C20R does not induce stress fiber formation, suggesting that ROCK-I but not PKN is required for stress fiber formation. These observations indicate that there are distinct interfaces in the effector domain of Rho for the different classes of effector proteins. Although at this time we cannot completely rule out the possibility that RB inhibits interaction of Rho with the other Rho targets such as PKN and p140mDia, RB may inhibit preferentially the interaction of Rho with Rho-kinase rather than with other Rho targets. On the other hand, C3 interferes with the whole functions of Rho. In fact, C3 inhibits the activity of PKN as well as Rho-kinase in Swiss 3T3 cells and the Rho-dependent recruitment of p140mDia to the dynamic membrane structures in Swiss 3T3 cells . C3 may inhibit the membrane ruffling by impairing the whole functions of Rho, whereas RB may inhibit the membrane ruffling by mainly (or specifically) impairing the function of Rho-kinase. We also found that not only C3 and RB/PH (TT) but also α-adducin-AA inhibited cell motility as well as membrane ruffling in NRK fibroblasts . This suggests that α-adducin is one of the substrates for Rho-kinase involved in cell motility, probably regulating the membrane ruffling in the leading edges of motile cells. As for the substrates of Rho-kinase, MLC is another major substrate which is thought to play an important role in cell motility. Indeed, injection of anti-MLC kinase antibody diminishes the cell motility of macrophages . Moreover, phosphorylated MLC is enriched in both the leading edges and rear ends of motile fibroblasts and epithelial cells , suggesting that a force derived from myosin-actin filament driven by the MLC phosphorylation contributes to cell motility. Thus, Rho, acting through Rho-kinase, appears to regulate cell motility through the spatial and temporal regulation of phosphorylation of certain substrates including adducin and MLC. It should be noted that in Swiss 3T3 or NIH3T3 fibroblasts, in which the dominant active Rac by itself is sufficient to induce the membrane ruffling , α-adducin phosphorylated at Thr445 was not observed in the dominant active Rac-induced ruffling areas, and neither C3, RB/PH (TT), nor HA-α-adducin-AA inhibited the dominant active Rac-induced ruffling (data not shown). On the other hand, we found that in KB epithelial cells as well as MDCK epithelial cells, α-adducin phosphorylated at Thr445 accumulated in the TPA-induced membrane ruffling areas, and that C3 and RB/PH (TT) inhibited the TPA-induced membrane ruffling (data not shown). When RB/PH (TT) or α-adducin-AA was injected to MDCK cells, the HGF-induced membrane ruffling was also inhibited as the TPA-induced membrane ruffling (data not shown). The regulatory system of the membrane ruffling may vary among cell types. It is conceivable that the membrane ruffling in a certain type of cell such as MDCK epithelial cells may require not only the Rac-mediated pathway but also the phosphorylation of adducin by the Rho/ Rho-kinase pathway, whereas that in other types of cells such as Swiss 3T3 fibroblasts may require only the Rac-mediated pathway. Further studies are necessary to determine what causes the differences between the two types of cell lines. These results indicate that the phosphorylation of α-adducin by the Rho/Rho-kinase pathway plays a crucial role in the regulation of membrane ruffling and cell motility. What is the function of the α-adducin phosphorylation in membrane ruffling and cell motility? Membrane ruffles contain complicated cytoskeletal structures composed of F-actin and other F-actin–associated proteins . Spectrin, which binds to F-actin beneath plasma membrane and forms a membrane cortical meshwork, also accumulates at the leading edges of motile cells . This lattice-like meshwork is dynamically reconstructed and required for the formation of membrane ruffling . Since the phosphorylation of α-adducin by Rho-kinase is known to promote the binding of α-adducin to F-actin and this in turn induces the recruitment of spectrin to F-actin , it is possible that the formation of a spectrin-F-actin meshwork is dynamically regulated in the active membrane ruffling by the Rho/Rho-kinase pathway . It is also possible that Rho-kinase modulates the F-actin–capping activity or cross-linking activity of adducin and then regulates actin dynamics in the membrane ruffling areas. Adducin is also a substrate of PKC, which is activated in response to TPA or HGF, and the phosphorylation of α-adducin by PKC inhibits the activity of adducin in promoting the formation of a spectrin– actin complex in vitro . TPA induces the phosphorylation of α-adducin at Ser726 and the redistribution of α-adducin phosphorylated at Ser726 from the cell membrane to cytoplasm accompanied by that of spectrin in renal proximal tubule epithelial and MDCK cells . Interestingly, α-adducin phosphorylated at Ser726 was also observed in the membrane ruffling areas (Matsuoka, Y., and V. Bennett, manuscript in preparation). It is conceivable that α-adducin phosphorylated by PKC dissociates from a spectrin-F-actin meshwork during membrane ruffling. Thus, it is tempting to speculate that the adducin function may be cyclically regulated through the phosphorylation by Rho-kinase and PKC during the action of TPA or HGF, and the alternative phosphorylation contributes to the turnover of a spectrin-F-actin meshwork for membrane ruffling. In this regard, it is interesting that α-adducin-DD inhibits the wound-induced migration of NRK49F cells but not their membrane ruffling. This suggests that the “on” and “off” states of α-adducin are necessary for cell migration . | Study | biomedical | en | 0.999994 |
10209030 | Leupeptin and trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E64) were obtained from Boehringer Mannheim . Ro 40-4388, Ro 61-7835, and Ro 61-9379 were provided by Dr. R.G. Ridley and Dr. R. Moon of Hoffmann LaRoche. Triethylamine (TEA), diethylamine (DEA), dibutylamine (DBA), 1-methyl-piperazine (1-MP) and dipropylamine (DPA) were purchased from Aldrich Chemical Co. Silicon oil was purchased from Dow Corning. 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethylester (BCECF-AM) was purchased from Molecular Probes Inc. [ 3 H]CQ (50.4 Ci per mmol) was purchased from DuPont NEN . [ 3 H]Hypoxanthine was purchased from Amersham and [ 3 H]amodiaquine (AQ; 106 mCi per mmol) was synthesized in house. All other reagents were purchased from Sigma Chemical Co. Parasites were cultured and synchronized using standard techniques . Drug sensitivity in the presence or absence of potential resistance reversers and hemoglobinase inhibitors was determined according to the method of Desjardins et al. . IC 50 values were calculated for each assay using the four parameter logistic method (Grafit program, Erithacus software; Kent Laboratories). The effect of the combination of CQ and Ro 40-4388 on parasite growth was tested by titration of the two drugs at fixed ratios proportional to their IC 50 values. The fractional inhibitory concentrations of the resulting IC 50 values were plotted as isobolograms . Erythrocytes infected with the K1 strain were synchronized and cultivated to the trophozoite stage and suspended in growth medium at a parasitemia of 10% and a hematocrit of 5%. At time 0, a sample was taken and the infected cells were separated, counted, and analyzed for hemoglobin and hemozoin as described below. The remaining cell suspension was incubated for 3 h at 37°C in the absence or presence of 10 μM Ro 40-4388, or 20 μM N- acteyl- l -leucyl- l -leucyl-methional (ALLM), or 20 μM N- acetyl- l -leucyl- l -leucyl-norleucinal (ALLN), or 20 μM leupeptin. After incubation, each was split into two aliquots. The infected cells from one aliquot were immediately separated from the uninfected cells using a Percoll–alanine gradient and counted as described . The hemoglobin and hemozoin concentrations of these cells were measured as described below. The infected cells from the remaining aliquot were washed three times in prewarmed complete medium and returned to culture for an additional 3-h incubation period. After incubation, the infected cells were separated, counted, and the hemozoin concentration was determined. Hemoglobin concentration was measured using Drabkins reagent as described . The hemozoin concentration in the pellet was determined after suspension in 2% (wt/vol) SDS in 0.1 M NaHCO 3 , pH 9.1, followed by centrifugation at 14,000 rpm for 5 min. The supernatant was discarded and this step repeated three times to remove all nonhemozoin heme. The remaining hemozoin pellet was dissolved in 1 ml of 0.1 M NaOH and the absorbance was read at 400 nm using a Beckman DU640 spectrophotometer. The amount of hemozoin/10 10 cells was calculated from a calibration curve of known amounts of FPIX dissolved in 0.1 M NaOH. Infected erythrocytes were suspended in the appropriate buffer containing [ 3 H]CQ, at a parasitemia of 1–2% and a hematocrit of 0.5%. At the required times, 100-μl samples were removed and the reaction was terminated upon centrifugation of the cells (14,000 rpm for 2 min) through silicon oil and processed for scintillation counting as described in Bray et al. . Parasite specific uptake was determined by subtracting counts because of an equal number of uninfected erythrocytes. Unless otherwise stated, CQ accumulation is expressed as the cellular accumulation ratio (CAR) which is the ratio of the amount of radiolabeled CQ in the parasites to the amount of radiolabeled CQ in a similar volume of buffer after incubation. The effect of the proteinase inhibitors Ro 40-4388, Ro 61-7835, Ro 61-9379, ALLM, ALLN, leupeptin, and E64, on the steady-state uptake of CQ was measured for 1 h at 37°C, in complete medium containing 1 nM [ 3 H]CQ. The reversibility of Ro 40-4388 (10 μM), ALLM (20 μM), and ALLN (20 μM) was determined as follows: [ 3 H]CQ was added at a concentration of 4 nM to inhibitor-treated and control groups. After 1 h, an aliquot was taken and processed for scintillation counting as described above. The remaining aliquot was washed three times in prewarmed medium (37°C). [ 3 H]CQ was re-introduced to the washed and control cells at a concentration of 4 nM. After 1 h, samples were removed and processed for counting. For the K1 isolate, parallel incubations were performed in the presence of 10 μM verapamil. In the case of ALLM and ALLN, inhibitors were added 45 min before the initial addition of CQ and a 10-min equilibration period was introduced after each wash. CQ–FPIX binding parameters, in the absence or presence of 10 μM Ro 40-4388, were determined as described in Bray et al. . For the K1 isolate, equilibrium binding parameters were obtained in sodium-free buffer (122.5 mM choline chloride, 5 mM KCl, 1.2 mM CaCl 2 , 0.8 mM MgCl 2 , 5.5 mM d -glucose, 1.0 mM K 2 HPO 4 , 10 mM Hepes, pH 7.4) in the absence or presence of 2 mM NH 4 Cl, 10 μM verapamil, or 100 nM monensin. Saturable uptake of CQ was calculated by subtracting nonsaturable CQ uptake from the total CQ uptake as described previously . Binding data are presented as Scatchard plots of saturable CQ uptake. The apparent K d of binding is given by the reciprocal of the slope and the amount of bound drug is given by the x-intercept. The effect of resistance modulators was determined over 1 h in growth medium containing 1 nM [ 3 H]CQ in the absence or presence of 10 μM verapamil, 100 nM nigericin, 100 nM monensin, 2 mM NH 4 Cl, 2 mM MTA, 2 mM TEA, 2 mM DEA, 2 mM DBA, 2 mM 1-MP, or 1 mM DPA. Care was taken to ensure the medium pH was 7.4 throughout. Optimum concentrations of these compounds were obtained in preliminary experiments by measuring the effect of serial 1:3 log dilutions of drugs on CQ uptake (data not shown). To determine the effect of bicarbonate on the uptake of [ 3 H]CQ, growth medium containing various concentrations of bicarbonate was shaken in atmospheric air at room temperature until the pH drift was complete. The pH was adjusted to 7.4 with 1 M HCl. Parasites were incubated in growth medium containing various concentrations of bicarbonate and 3 nM [ 3 H]CQ for 1 h and terminated thereafter. Parasites were isolated from the host cell by selective lysis of the host cell compartment . Ringer's buffer modified to simulate an intracellular milieu was used throughout . In selected experiments, sodium chloride in the buffer system was replaced with molar equivalents of either choline chloride or N -methyl- d -glucamine chloride. CQ uptake was determined by suspending the cell pellet containing purified free parasites, in 200 μl of the appropriate buffer containing 50 nM [ 3 H]CQ, prewarmed to 37°C. Drug uptake was determined in the absence or presence of 100 μM 5- N -ethyl- N -isopropyl amiloride (EIPA) or 10 μM verapamil. At the appropriate time points, aliquots were removed and the reaction was terminated by centrifugation (14,000 rpm, 1 min, at 4°C). The sample was placed in an ice-bath, the supernatant was removed, and the pellet was washed once in the appropriate ice-cold buffer without [ 3 H]CQ. After centrifugation (14,000 rpm, 1 min, at 4°C), the supernatant was carefully removed with a drawn out glass Pasteur pipette and the pellet was processed and counted for infected erythrocytes. CQ uptake into isolated parasites was ∼30% of intact infected erythrocytes' uptake with over 30 min of CQ exposure. To investigate whether this reduced CQ uptake occurs because of impaired NHE activity, we compared the cytosolic pH of isolated parasites to that of parasites within intact host erythrocytes. In agreement with previous reports , we found no significant differences in cytosolic pH in these preparations. For example, the mean cytosolic pH for the K1 clone in its host cell was 6.902 (SEM = 0.045, n = 12) compared with 6.992 (SEM = 0.038, n = 22) when the parasite is liberated from the host cell. In addition, free parasites exhibited a marked cytosolic acidification in sodium-free buffer (0.76 pH unit ± SEM 0.045, n = 6), in full agreement with previous reports . Thus, NHE is functional in isolated parasites. We also investigated the possibility that the reduced CQ uptake occurs because of a loss of viability. Isolated parasites were found to incorporate radiolabeled hypoxanthine and isoleucine at the same rate as intact infected erythrocytes for at least 6 h (data not shown). Since the parasites are viable and NHE is functioning normally, we assumed that the reduced accumulation of CQ happens because of cessation of hemoglobin trafficking from the host cell at time 0 that reduces the amount of FPIX available for CQ binding. Measurement of FPIX polymerization inhibition by amiloride analogues and proteinase inhibitors employed a modification of the procedure described by Raynes et al. . An aliquot (100 μl) of trophozoite lysate and FPIX (100 μl of 3 mM in 0.1 M NaOH) was mixed with an aliquot of 1 M HCl (10 μl) and sodium acetate (500 mM, pH 5.2) to give a volume of 900 μl in each tube. A series of drug concentrations was prepared in ethanol and 100 μl of each was added to the appropriate samples. The effect of ethanol on the polymerization process was assessed by adding 100 μl of ethanol to the control samples. Samples were mixed and incubated for 12 h, with occasional mixing. After incubation, samples were centrifuged (14,000 rpm, 15 min, at 21°C) and the hemozoin pellet repeatedly washed with 2%(wt/vol) SDS in 0.1 M sodium bicarbonate, pH 9.0, with sonication (30 min, at 21°C; FS100 bath sonicator; Decon Ultrasonics Ltd.) until the supernatant was clear (usually 3–4 times). After the final wash, the supernatant was removed and the pellet was resuspended in 1 ml of 0.1 M NaOH, incubated for an additional 1 h at room temperature. Afterwards, samples were mixed with a pipette. The hemozoin content was determined by measuring the absorbance at 400 nm (Beckmann DU640 spectrophotometer) using a 1-cm quartz cuvette. The amount of hemozoin formed during incubation was corrected for preformed hemozoin (the amount of preformed hemozoin in the parasite extract was determined from a sample containing extract, but no substrate, which was incubated and repeatedly washed with 2% SDS as previously stated). The concentration of drug required to produce 50% inhibition of polymerization (IC 50 ) was determined graphically as described for the drug sensitivity assays. Erythrocyte ghost membranes were prepared as described previously . Membranes (0.27 mg protein) were loaded with FPIX (2–5 μM) in 0.2 M Hepes buffer, pH 7.0 (HB7) at 37°C for 7 min followed by centrifugation (14,000 rpm, 10 min). The supernatant was discarded and the pellet was washed once in HB7. Samples of FPIX-loaded membrane (0.01 mg protein) were suspended in 1 ml of HB7 containing 50 nM [ 3 H]CQ and incubated in the absence or presence of various concentrations of the proteinase inhibitors, amiloride and the amiloride analogues EIPA, 5- N - N -hexamethylene amiloride (HMA), and 5- N -isobutyl- N -methyl amiloride (IBMA) for 10 min at 37°C. The membrane suspension was centrifuged (14,000 rpm, 2 min), the supernatant removed, and the pellet washed once in ice-cold HB7 without [ 3 H]CQ. The remaining pellet was solubilized and processed for counting, as described above. To measure CQ–FPIX binding affinity in a parasite-free system, the membranes were loaded in HB7 with 2 μM FPIX as above and samples (0.01 mg protein) were incubated in 1 ml HB7 containing 3 nM [ 3 H]CQ and 5–10 μM nonradioactive CQ for 10 min at 37°C. The reaction was terminated by centrifugation and samples were processed as above. Nonspecific CQ binding was estimated from parallel incubation of FPIX-free membranes and subtracted from the total binding for each CQ concentration. Binding affinity was estimated by a computer fit of the data to the Michaelis-Menten equation. The displacement of preaccumulated [ 3 H]CQ from parasite debris by EIPA was measured as described previously . Before lysis, erythrocytes infected with the CQS (HB3 strain) were loaded with 50 nM [ 3 H]CQ in complete medium for 30 min at 37°C. Pure isolated food vacuoles were prepared by modifying the methods of Goldberg et al. and Saliba et al. . Suspensions of synchronized trophozoites of the HB3 strain (∼15% parasitemia) were washed three times in PBS, pH 7.4. Each 5-ml sample of washed cell-pellet was resuspended in PBS containing 0.15% saponin, incubated for 5 min, and centrifuged (4,500 rpm for 5 min). The isolated trophozoites were washed repeatedly in ice-cold PBS until the supernatant was clear. The trophozoite pellet was collected and resuspended in 10 vol of ice-cold trituration buffer (0.25 M sucrose, 10 mM sodium phosphate, 0.5% streptomycin sulfate, pH 7.1) and triturated three times on ice, using a 27-gauge 3/4 in needle. The suspension was centrifuged (13,000 rpm, 2 min, at 4°C), supernatant discarded, and the pellet resuspended in 5 vol of buffer (2 mM magnesium sulfate, 100 mM potassium chloride, 10 mM sodium chloride, 25 mM Hepes, 25 mM sodium bicarbonate, 5 mM sodium phosphate, pH 7.1). To each 1 ml of the suspension 20 μl of 5 mg/ml DNaseI was added and the suspension was incubated at 25°C for 5 min, followed by centrifugation (13,000 rpm, 2 min, at 4°C). The supernatant was discarded and the pellet was resuspended in 5 vol of ice-cold trituration buffer and triturated once. The suspension was layered on top of 7 ml of ice-cold 42% Percoll solution containing 0.25 M sucrose, 1.5 mM magnesium chloride, pH 7.1, and centrifuged (12,000 rpm, 30 min, at 4°C). After centrifugation, the purified food vacuoles were harvested from the bottom of the gradient and washed three times in ice-cold buffer (0.25 M sucrose, 10 mM sodium phosphate, 1.5 mM magnesium chloride). Purity was checked by electron microscopy and by using assays of host cell and parasite cytosol marker enzymes as described previously . Electron microscopy revealed no contamination with other organelles or membranes and ∼50% of the vacuoles had intact membranes (data not shown). Contamination with the parasite cytosolic enzyme lactate dehydrogenase was <0.7%. Compared with isolated trophozoites and contamination with host cell acetylcholine esterase was below the limits of detection in the assay. Pellets of pure food vacuoles were added to 20 vol of ice-cold 500 mM sodium acetate, pH 5, and immediately subjected to five cycles of rapid freezing in liquid nitrogen and thawing at room temperature. The suspension was triturated 10 times with a 27-gauge needle and centrifuged (13,000 rpm, 3 min, 4°C). The proteinase-rich supernatant was collected and used for the cell-free assay. Protein content was measured using the bicinchoninic acid assay . Samples of the enzyme extract (2–10 μl) were added to 200 μl of 500 mM sodium acetate, pH 5, containing [ 3 H]CQ or [ 3 H]AQ. 100 μl of a 50-μM solution of human hemoglobin was added to the mixture. Purified erythrocyte ghost membranes (0.01 mg protein) were added to act as carriers for the CQ–FPIX complex. The samples were incubated for 1 h at 37°C in the absence or presence of 10 μM Ro 40-4388, or 20 μM Ro 61-7835, or 20 μM Ro 61-9379, or 20 μM leupeptin, or 20 μM E64. After incubation, the samples were centrifuged (14,000 rpm, 1 min) and the supernatant was removed gently with a drawn out glass Pasteur pipette to avoid disturbing the pellet. The pellet was washed once in ice-cold sodium acetate buffer without radiolabel and the remaining pellet was solubilized and processed for counting as described above. Parasite cytosolic pH was estimated using BCECF-AM as described by Wünsch et al. . The parasite suspension preloaded with BCECF was incubated at 37°C for 15 min in a perfusion chamber and the cells were allowed to settle on a glass coverslip coated with poly- l -lysine. The experimental chamber was transferred to the stage of an inverted Diaphot microscope ( Nikon ). At appropriate time points, 10 μM Ro 40-4388, or 2 mM NH 4 Cl, or 2 mM methylamine was added to the perfusion buffer and the pH was monitored. NHE activity in sodium-free buffer was monitored by measuring the ability of the cell to recover from an acid load: the cells were perfused with Ringer's buffer containing 40 mM NH 4 Cl, after which the perfusion buffer was changed to Ringer's with one molar equivalent of choline replacing sodium. When the fall in cytoplasmic pH stabilized, the perfusion buffer was changed to sodium Ringer's to allow the cytosolic pH to recover. Digital imaging microfluorimetry was carried out with an image analysis system (Quanticell 700 series; Applied Imaging International Ltd.) incorporating an intensified camera (CCD; Photonic Science). Background subtraction was performed independently for each excitation wavelength used. The autofluorescence of unloaded parasites was negligible. The excitation wavelengths used were 440 and 490 nm with emission measured above 510 nm. Calibration was performed for each cell using the nigericin/ high K + method that uses two or three buffers of known pH . Since no ultrastructural detail could be observed under light microscopy, other than the food vacuole, the reported pH values are average values for the entire parasite cytosol minus the food vacuole. We determined the effect of a range of proteinase inhibitors on CQ uptake. In addition to Ro 40-4388, two other specific plasmepsin inhibitors, Ro 61-7835 and Ro 61-9379, inhibited CQ uptake at low micromolar concentrations . Similar inhibition was observed with the tripeptide aldehydes, ALLM and ALLN, which are known inhibitors of the processing of proplasmepsins I and II into active mature enzymes . The other cysteine proteinase inhibitors tested, E64 and leupeptin, were much less effective. Inhibition of proplasmepsin processing by the tripeptide aldehydes and the direct inhibition of the plasmepsins with Ro 40-4388 are reversible . We have established that the inhibition of CQ uptake by both classes of compounds is also reversible, as predicted by our hypothesis . In CQS parasites, CQ uptake is inhibited almost completely by the proteinase inhibitors . In CQR parasites, the effect was smaller but the inhibition of the resistance reversing effect of verapamil was spectacular . This observation is in agreement with our hypothesis that verapamil increases CQ uptake by increasing the affinity of CQ–FPIX binding . Our data show that CQ–FPIX binding determines the amount of drug that is taken up by the parasite. The marked antagonism of CQ activity in the presence of Ro 40-4388 suggests that binding to FPIX also determines the antimalarial activity of CQ. Our model of the mechanism of CQ uptake is supported by the demonstration that proteinase inhibitors efficiently inhibit the degradation of hemoglobin and liberation of FPIX under the same conditions used to inhibit CQ uptake . Hemoglobin digestion is almost completely inhibited by ALLM, ALLN, and Ro 40-4388. On the other hand, the effect of leupeptin was smaller. It is very difficult to accurately measure the concentration of free FPIX in parasites, so we have measured the concentration of hemozoin (polymerized FPIX). At the concentrations used, none of the compounds had any direct effect on the polymerization of FPIX in vitro (data not shown). However, all four compounds inhibited the production of hemozoin by intact parasites to some extent . These data show that the concentration of FPIX is reduced when hemoglobin digestion is blocked. The inhibition was much more pronounced with ALLM, ALLN, and Ro 40-4388 than with leupeptin and was found to be reversible. Hemozoin production resumed at the normal rate when the proteinase inhibitors were removed by washing . All these results are in agreement with the effect of the same inhibitors on CQ uptake and provide compelling evidence that CQ uptake results from binding of the drug to FPIX generated inside the infected erythrocyte. The final piece of experimental evidence that proves the central role of FPIX binding in the uptake of CQ was obtained using a cell-free system. The binding characteristics of intact CQS parasites can be reconstituted using an erythrocyte ghost membrane preparation preloaded with FPIX. The capacity (B max ) of saturable CQ binding to these membranes was dependent on the FPIX concentration, although at higher loading concentrations the apparent binding affinity was reduced due to aggregation of FPIX (data not shown). We measured an affinity ( K d ) for this interaction of 25 nM . This value is the same as the apparent K d of saturable CQ uptake in CQS P . falciparum , again indicating that FPIX binding is all that is required to account for the saturable uptake of CQ . Other experiments indicate that CQ binding sites can be generated from native hemoglobin by an extract from purified food vacuoles . There is little doubt that CQ binding is absent when either hemoglobin or vacuole extract is omitted, or when the extract has been heat-treated (data not shown). The same proteinase inhibitors that block the uptake of CQ into intact cells block CQ binding in the cell-free system . In fact, the percent inhibition of CQ binding in the cell-free system is almost perfectly correlated with the inhibition of CQ uptake into intact cells by similar concentrations of these compounds (R 2 = 0.99, P < 0.001). AQ binding sites can be generated in a similar fashion and have a similar inhibitor profile . These data suggest that the mechanism of AQ uptake is the same as that of CQ, i.e., the uptake of both drugs is driven by their binding to FPIX. The initial rate of uptake of CQ into CQS parasites is rapid, prompting some authors to suggest that CQ is actively transported . However, our data suggest that the initial rate of CQ uptake is determined by the intraparasitic binding of CQ to FPIX rather than active transport. We found that after only a 5-min exposure of HB3 (CQS) parasites to CQ, the number of CQ binding sites (B max ) was reduced from 17.2 μmol/liter to 6.9 μmol/liter by the specific hemoglobinase inhibitor Ro 40-4388 . In contrast to our explanation, others believe that CQ is transported through the NHE of CQS parasites . The NHE of CQR isolates was activated constitutively, incapable of CQ transport, and responsible for an elevated cytoplasmic pH . We were not able to reproduce these observations. In our hands, cytosolic pH is subject to a greater degree of variation and is actually lower in the resistant isolate (HB3, pH 7.183 ± SEM 0.04, n = 17; K1, pH 6.902 ± SEM 0.046, n = 12). The reason for this discrepancy is unknown but there may be a methodological basis: our pH measurements are average values for the whole cytosol (minus the food vacuole), whereas those of Wünsch et al. refer to smaller regions of interest within the cytosol. Elevated cytoplasmic pH of CQR isolates is a fundamental tenet of the NHE hypothesis that probably does not apply to the isolates used in this study. Nonetheless, we have assessed the likely impact of small variations in cytosolic pH on the binding of CQ to FPIX. We found that the binding of CQ to FPIX in ghost membranes is sensitive to buffer pH, but this effect is probably not significant across the range of reported cytosolic pHs . We have also tested the effect of hemoglobinase inhibition on cytosolic pH although such an effect seems unlikely. We found that concentrations of Ro 40-4388, sufficient to block CQ uptake, had no effect on cytosolic pH, indicating that blocking hemoglobin degradation does not affect the cytosolic pH and that this compound does not block CQ uptake by interacting with the parasite NHE . It is proposed that CQ is driven through the NHE of CQS parasites in a rapid burst of sodium-proton exchange stimulated by CQ itself . We tested this proposal directly by replacing the sodium ions in the media with nonexchangeable cations and monitoring the uptake of CQ into isolated parasites. This maneuver resulted in a marked inhibition of the NHE of isolated parasites, as evidenced by the inability to recover from an acid load (0.76 pH unit ± SEM 0.045, n = 6). It follows that sodium-free buffer should produce a very marked effect on CQ uptake if the NHE is involved in this process. It was observed that the initial rate of CQ uptake and steady-state CQ accumulation into isolated CQS parasites (HB3 strain) remained constant regardless of the concentration of sodium ions in the buffer . These data demonstrate that CQ uptake is independent of the parasite's NHE activity status. The reported ability of CQ to stimulate the NHE of CQS parasites is interesting but it appears to have no involvement in the mechanism of CQ uptake. We also tested the effect of the sodium ion concentration on the uptake of CQ into isolated CQR parasites (K1 strain). The results, presented in Fig. 6 C, again show that CQ uptake is unchanged when sodium ions are removed from the buffer. In addition, the reversal of CQ resistance by verapamil was independent of NHE activity, as it is retained in sodium-free medium . CQ accumulation of CQR parasites is reduced by a similar ratio compared with CQS parasites regardless of the sodium ion concentration. Moreover, it is independent of the presence or absence of the host cell . Therefore, all of the phenotypic features of CQ uptake that distinguish CQR parasites from CQS parasites are retained in sodium-free medium thereby providing compelling evidence that the parasite NHE is not involved in CQ resistance. EIPA significantly inhibits the uptake of CQ even though there are no sodium ions in the buffer and NHE is inactive . These results are interesting because they indicate that EIPA inhibits CQ uptake by a mechanism distinct from the inhibition of NHE. One possibility is that EIPA inhibits the binding of CQ inside the parasite. This assessment is supported by the data presented in Fig. 7 , showing EIPA displacing prebound CQ from parasite debris in a concentration-dependent manner. This interaction is not due to displacement of CQ bound to the NHE protein as this could only account for ∼5% of the measured CQ binding . Amiloride and its derivatives were shown to competitively inhibit the saturable uptake of CQ . Since we attribute saturable uptake of CQ to CQ–FPIX binding, we have looked for an interaction of these compounds with FPIX. Such an interaction is indicated by the data in Fig. 8 , showing amilorides inhibiting FPIX polymerization. Indeed, HMA is almost as efficient an inhibitor of FPIX polymerization as CQ itself. These data suggest that amiloride derivatives might inhibit the uptake of CQ into P . falciparum by preventing CQ–FPIX binding. We tested this possibility directly and demonstrated that these compounds inhibit CQ–FPIX binding in loaded ghost membranes in a dose-dependent manner . Interestingly, the 50% inhibitory concentration for EIPA was within the range reported for 50% inhibition of CQ uptake into P . falciparum by EIPA . Furthermore, the rank order of inhibition of CQ–FPIX binding by the amiloride analogues (HMA > EIPA > IBMA ≫ amiloride) is the same as the rank order of their inhibition of CQ uptake into intact parasitized erythrocytes . These data indicate that amiloride analogues do inhibit CQ uptake by blocking CQ–FPIX binding rather than inhibiting the parasite NHE. We also hypothesized that verapamil reverses CQ resistance by increasing the apparent affinity of CQ binding to FPIX . If our working model is correct and if EIPA binds to FPIX at the expense of CQ, then EIPA should ablate the verapamil effect; this is clearly demonstrated in Fig. 6 C. Although CQ is not actively transported through NHE it is still possible that CQ resistance may arise from changes in the cytosolic pH . Accordingly, we examined the effect of various agents known to perturb intracellular pH on the uptake of CQ by CQS and CQR parasites. We found that ammonium chloride, methylamine, a variety of alkylamines, and the carboxylic ionophores, monensin and nigericin, selectively increase the CQ accumulation of CQR parasites. The maximum effect was found at concentrations of 1–2 mM for amines and 100 nM for ionophores. In contrast, CQ accumulation by CQS parasites was either unaffected or reduced . In this respect, the amines and ionophores mimic the effect of verapamil . Ammonium chloride and methylamine were also capable of reversing CQ resistance in the same way as verapamil (although the effect was not as pronounced). The remaining weak bases could not be assessed for their resistance reversal potential because of their inherent cytotoxicity. Ammonium chloride, methylamine, and verapamil were selected to investigate the possibility that resistance reversers alter cytosolic pH of CQR parasites. We found that these compounds had no significant effect on cytosolic pH at the low concentrations required to reverse resistance (data not shown). Therefore, the ability of these compounds to increase CQ accumulation is unlikely caused by any interaction with cytosolic pH regulators like NHE. Indeed, any possible contribution of the NHE in the process of resistance reversal is negated by the data showing that ammonium chloride, monensin, and verapamil all increase the apparent affinity of CQ binding to FPIX in sodium-free medium, when the NHE is inactivated . We were also interested to see if any other cytosolic pH regulators could influence CQ uptake. In common with other eukaryotic cells, it is possible that P . falciparum parasites possess a chloride/bicarbonate exchange mechanism . If so, and if this protein can influence CQ uptake as suggested , then CQ uptake should be influenced by the bicarbonate concentration of the medium. It was found that CQ uptake into CQS and CQR parasites was not effected by the concentration of bicarbonate in the medium . Taken together, our data suggest that none of the major cytosolic pH regulators play a major role in the uptake of CQ or in the mechanism of CQ resistance. The specificity of antimalarial activity of CQ stems from the potential of malaria parasites to accumulate more CQ than any other type of eukaryotic cell. CQR parasites of some species, including P . falciparum , have evolved ways to reduce the extent of CQ accumulation. A better understanding of the mechanism of CQ uptake in P . falciparum would be a significant step forward since it should explain the specificity of the drug and may also offer clues to the basis of resistance. CQ undoubtedly penetrates the parasite by passive diffusion as observed in other eukaryotic cells . CQ is a diprotic weak base and, as such, undoubtedly accumulates to some extent inside the acidic compartments of the parasite by a proton trapping mechanism . This mechanism may account for a considerable concentration of CQ inside cells . However, it probably does not account for the full extent of CQ accumulation by P . falciparum , which is notably greater than CQ accumulation by other eukaryotic cells with large acidic compartments . Pioneering studies by Fitch in the 1970s demonstrated that CQ uptake into P . falciparum is saturable, energy-dependent, and possibly inhibited by various compounds . These studies suggest that malaria parasites possess an additional CQ concentrating mechanism acting in concert with passive diffusion and proton trapping. Two putative CQ concentrating mechanisms are currently under consideration: active CQ import mediated by the parasite NHE ; and intracellular binding of CQ to FPIX . The NHE hypothesis initially appears plausible and many of the supporting data are robust. However, a close scrutiny of the available literature reveals some intriguing inconsistencies that call into question the mechanistic explanation offered by Wünsch et al. . Particularly problematic is the uptake of AQ, a close structural analogue of CQ. Early studies showed that AQ competitively inhibits the uptake of CQ in P . falciparum , suggesting that the same mechanism drives the uptake of both drugs . We reported that the uptake of AQ into CQR parasites is equivalent to the uptake of CQ into CQS parasites . It is hard to see how the extensive uptake of AQ into CQR parasites can be driven by an NHE that apparently is activated constitutively and incapable of drug transport . A further difficulty is encountered when the effect of verapamil is considered. Many studies have highlighted the ability of verapamil to selectively increase the uptake of CQ into CQR parasites . Furthermore, this property of verapamil was linked perfectly to CQ resistance in the progeny of a genetic cross of CQR and CQS clones . If NHE is responsible for CQ uptake, then verapamil would somehow have to selectively stimulate NHE of resistant parasites that is constitutively activated and incapable of CQ transport. However, this is an unlikely scenario, doubly so, when one considers the large quantity of literature demonstrating verapamil functioning to inhibit rather than stimulate drug transporters and ion channels . The active transport model is inconsistent with our own data showing a role for CQ–FPIX binding in the uptake of CQ, in verapamil-sensitive CQ resistance and in the uptake of AQ . We rigorously tested these two hypotheses and found that the FPIX binding model remains valid, whereas the altered NHE model failed. We have tested the assertion that a rapid burst of sodium–proton exchange is required to drive uptake of CQ into CQS parasites. We used isolated parasites for these experiments and assumed that NHE activity is not impaired under such conditions. We show here as others have shown that parasitic NHE is fully functional after the parasite has been isolated from its host cell . We found that replacing sodium ions in the buffer with nonexchangeable cations, such as choline or N -methyl- d -glucamine, inactivated NHE but had no effect on the amount of CQ taken up at steady-state by isolated CQS parasites . Moreover, sodium-free buffer had no effect on the initial velocity of CQ uptake into CQS parasites . This was measured over the first 5 min after the addition of CQ, which coincides with the reported period of maximal stimulated NHE activity . Since there can be no rapid burst of sodium–proton exchange in sodium-free buffer and CQ uptake is not altered by these conditions, our results strongly suggest that any ability of CQ to stimulate the NHE of CQS parasites is unrelated to the mechanism of CQ uptake. In addition, we found that stimulation of CQ uptake into CQR parasites, produced by verapamil and other resistance reversers, was unrelated to the activity of the NHE because these effects are retained in sodium-free buffer . Consequently, the effect of verapamil on the uptake of CQ cannot be attributed to modulation of NHE activity via the calcium-calmodulin regulatory pathway, as postulated by Sanchez et al. . Hence, the clone-specific phenotypic characteristics of CQ uptake into CQS and CQR parasites are retained in conditions which completely inactivate the NHE . Therefore, it is difficult to see how these characteristics can be related in any way to the activity of the parasite NHE. We have demonstrated that amiloride analogue inhibition of CQ uptake occurs because of inhibition of CQ–FPIX binding by these compounds, rather than the inhibition of the parasite NHE. In a critical series of experiments, we were able to demonstrate that the inhibition of CQ uptake by EIPA was distinct from any activity of this drug against NHE. CQ uptake is undiminished in sodium– free buffer when NHE is inactive, yet it is effectively inhibited when EIPA is present in this buffer . These data strongly suggest that blocking CQ uptake by EIPA is caused by inhibition of a process that does not require sodium–proton exchange for CQ uptake. This directly violates the fundamental requirement of the NHE hypothesis. There is solid experimental support for the FPIX model . Therefore, the demonstration that EIPA displaces prebound CQ from parasite debris , binds to FPIX , and inhibits the binding of CQ to FPIX-loaded ghost membranes provides direct evidence that EIPA inhibits CQ uptake into parasites by binding to FPIX. Furthermore, the rank order of activity of the amiloride analogues (HMA > EIPA > IBMA ≫ amiloride) is the same for the inhibition of CQ–FPIX binding as it is for the inhibition of CQ uptake . Examination of the chemical structure of amiloride reveals that either of the two terminal amino groups of the guanidine function has the potential to coordinate with the iron center of the porphyrin as an axial ligand . Hoe 370, a specific NHE inhibitor that is structurally unrelated to the amiloride analogues, also has been shown to inhibit CQ uptake . Note, this compound also contains a guanidine function and may also bind to FPIX. Inhibition of CQ uptake by these specific NHE inhibitors provided the best evidence of active import of CQ through the NHE. However, in the light of the data reported here, we believe that this property of NHE inhibitors must now be considered to support the alternative theory that CQ uptake is governed by its binding to FPIX. The central theme of the studies presented here is the definitive proof, after some thirty years of controversy, that saturable CQ uptake is driven by its binding to FPIX. We recently demonstrated that Ro 40-4388, a potent and specific inhibitor of the parasite proteolytic enzyme plasmepsin I , produces a concentration-dependent reduction in the number of CQ binding sites of intact parasitized erythrocytes . Here we have extended this observation blocking hemoglobin degradation by two distinct mechanisms. The plasmepsins are thought to initiate hemoglobin degradation by cutting the Phe33-Leu34 bond of the alpha chain. This unfolds the hemoglobin tetramer, allowing further proteolysis and the release of FPIX . Although the parasite contains other hemoglobinase enzymes, there is good evidence that the inhibition of plasmepsin I alone is sufficient to stop the digestion of hemoglobin and release of FPIX . We have used Ro 40-4388, Ro 61-7835, and Ro 61-9379 to block hemoglobin cleavage. All three compounds are potent inhibitors of the parasite aspartic hemoglobinase enzymes, plasmepsin I and plasmepsin II . In addition we have prevented proplasmepsin processing with ALLM and ALLN. We provide evidence that the reversible inhibition of CQ binding produced by Ro 40-4388 and other proteinase inhibitors stems from a reversible cessation of hemoglobin digestion and FPIX release in the parasite . Perhaps the most convincing support for our hypothesis comes from the demonstration that a purified food vacuole extract can generate CQ binding sites in a cell-free system using human hemoglobin as a substrate . The correlation of the inhibitor specificity of this process with that of CQ uptake into intact cells is compelling. Nevertheless, it is important that any proposed mechanism accounts quantitatively as well as qualitatively for CQ uptake. Further analysis of the data presented in Fig. 3 B reveals that this is the case. The extract from 10 6 vacuoles generates 123.72 fmol of CQ binding sites. At the external CQ concentration used (0.61 nM) this equates to a CAR of 2,399 (assuming one food vacuole per infected erythrocyte). This figure compares with a CAR of ∼2,600 for intact parasites under similar conditions . Much proteolytic activity has undoubtedly been lost in our assay because of poor enzyme extraction efficiency and leakage of enzymes from vacuoles during purification. Therefore, intact parasites clearly possess more than enough proteolytic activity to account for the uptake of CQ. FPIX–CQ binding is the principal driving force for drug uptake including the initial CQ uptake kinetics, measured after 5 min exposure to CQ and previously attributed to a carrier-mediated import mechanism . Published estimates of the rate of hemoglobin digestion adequately defend this argument. It is estimated that each parasite degrades 0.06 fmol of hemoglobin per hour . This would liberate 50 μmol FPIX per liter of parasites in 5 min, i.e., more than enough to account for initial rate of bound CQ even at a stoichiometry of 2 FPIX:1 CQ. If so, this indicates that CQ uptake may limit itself by stopping hemoglobin digestion since the steady-state B max is only 30–40 μmol per liter . CQ uptake might be limited by CQ–FPIX inhibition of the hemoglobinase enzymes. It is possible that sufficient CQ–FPIX complex remains within the vacuole to inhibit the proteolytic enzymes . To quote Chou et al. , “Unequivocal identification of an isolated substance as a drug receptor requires (a) that affinities and specificities of binding of the drug match those of the receptor, (b) that the drug is ineffective when the putative receptor is absent from the organism, and (c) that drug effectiveness returns when the receptor is reintroduced into the organism.” The specificity of hemoglobinase inhibitors that inhibit cellular CQ uptake is identical to their specificity in the cell-free enzymatic CQ binding assay . These drugs are specific and reversible inhibitors of FPIX generation . This biochemical knockout has permitted us to show that FPIX can be reversibly removed, causing a reversible inhibition of drug uptake . In addition to governing the uptake of the drug, CQ–FPIX binding determines antimalarial activity since the combination of CQ and hemoglobinase inhibitors is markedly antagonistic . Furthermore, we were able to match the affinity of binding of CQ to CQS parasites to the affinity of CQ–FPIX binding in a parasite-free system . Thus, all of the above criteria have been satisfied and identify FPIX as the CQ receptor in P . falciparum . Our data indicate that NHE has no involvement in the mechanism of CQ resistance . Indeed, any involvement of cytosolic pH regulation seems unlikely. Instead, our data suggest that CQ resistance stems from an alteration in the local environment of FPIX generation in acid vesicles. We found that a wide range of lysosomotropic compounds mimics the effects of verapamil . This could indicate the inhibition of a drug transporter similar to P-glycoprotein. However, since many of these compounds are not known to interact with P-glycoprotein, we suggest an alternative mode of resistance reversal. The concentrations required to reverse resistance produced no alkalinization of the parasite cytosol but might be expected to produce a significant alkalinization of lysosomes and endosomes . There is evidence in the literature that hemoglobin digestion begins in hemoglobin delivery vesicles, before they fuse with the food vacuole . To protect the parasite, the resistance mechanism must be operational throughout the endocytic pathway. The intracellular localization of CQ resistance gene CG2 throughout the endocytic pathway is certainly consistent with this hypothesis . It is our belief that CQ resistance results from a selective change in vesicular function within relevant hemoglobin processing acidic compartments. This reduces the affinity of CQ–FPIX binding that can be reversed by lysosomotropic agents. CG2 and related proteins could potentially alter the binding of CQ to FPIX by directly binding to FPIX or by altering vesicular pH or buffering capacity. All of these mechanisms could be modulated by vesicle alkalinization and are currently under investigation in our laboratory. | Other | biomedical | en | 0.999996 |
10209031 | Cell culture reagents were purchased from Gibco Laboratories . cDNAs for human wild-type Fyn, G 2 A-Fyn, and C 3,6 S-Fyn ( 1 ) and NMT ( 28 ) and rabbit polyclonal antiserum raised against Fyn SH43 protein ( 3 ) were from lab stocks. pEFBOS-CD8-ζ, CD8-ζT76, and CD8-ζ4F ( 16 , 22 ) were kindly donated by Dr. Arthur Weiss (Department of Immunology, University of California, San Francisco, San Francisco, CA). pSVΔSH2-Fyn, containing mutant Fyn lacking amino acids 144–248, was kindly provided by Drs. Nicolas Dunant and Kurt Balmer-Hofer (Friedrich Miescher Institute, Basel, Switzerland). pEGFP-N1 and rabbit polyclonal antibody against green fluorescent protein (GFP) were obtained from Clontech . Anti–CD8 monoclonal antibody solution and high purity digitonin were from Calbiochem . Protein A–agarose and protein A/G + agarose beads and antiphosphotyrosine (PY99) monoclonal antibody solution were purchased from Santa Cruz Biotechnology . dl -α-Hydroxymyristic acid (2-hydroxytetradecanoic acid), defatted BSA, and filipin were from Sigma Chemical Co. and n -octylglucoside was from Boehringer Mannheim . Tran 35 S-label and 125 I-NaI were from DuPont-NEN . Synthesis and radioiodination of IC16 (16-iodohexadecanoic acid) and IC13 (13-iodotridecanoic acid) fatty acid analogues were performed as previously described ( 2 ). Construction of plasmids containing wild-type Fyn, G 2 A-Fyn, C 3,6 S-Fyn, and Gα o -Fyn was described before ( 1 , 41 ). For generation of K 7 A-, K 9 A-, K 7,9 A-Fyn, and Lck(10)Fyn constructs, sense oligonucleotides were synthesized to encode the upstream region of pGEM3Z, an NcoI site, and either the first 11–14 amino acids of human Fyn, containing lysines to alanine substitutions at positions 7 and/or 9, or the first 10 amino acids of human Lck, followed by amino acids 11–16 of human Fyn. An antisense oligonucleotide was used corresponding to a region of the SH3 domain of Fyn ( 1 ). Sense and antisense primers were used with pGEM3Z-Fyn as a template to generate mutant cDNAs, which were digested with NcoI and BstXI to produce 76-bp fragments that were used to replace the corresponding fragments of Fyn in pSP65. A G 2 A-FynKRas construct was generated by PCR using a sense oligonucleotide encoding nucleotides 1110–1130 of human Fyn containing a unique BglII site, and an antisense oligonucleotide corresponding to the last 18 nucleotides of Fyn, followed by the last 54 nucleotides of KRas4B, a stop codon, and a SalI site. The primers were used with pGEM3Z-Fyn as a template. The PCR product was digested with BglII and SalI to produce a 500-bp fragment that was used to replace a corresponding carboxy-terminal fragment of G 2 A-Fyn in pSP65. All Fyn constructs in pSP65 were subsequently digested with EcoRI and SalI, and ligated into pCMV5. FynKRas was subsequently generated by digestion of pCMV5-G 2 A-FynKRas with BglII and SalI, followed by ligation into BglII and SalI cut pCMV5-Fyn. G 2 A,C 3 S-FynKRas was generated by PCR, with pGEM3Z-Fyn as a template, using a sense oligonucleotide encoding an NcoI site followed by the first eight amino acids of human Fyn, containing a glycine-to-alanine substitution at position 2 and a cysteine-to-serine substitution at position 3, and an antisense oligonucleotide corresponding to the SH3 domain of Fyn ( 1 ). The PCR product was digested with NcoI and BstXI to generate a 76-bp fragment that was used to replace the corresponding fragment of pSP65-FynKRas. G 2 A,C 3 S-FynKRas subcloned into pCMV5 as described above. ΔSH2-Fyn was subcloned into pSP65 by digestion of pSVΔSH2-Fyn with BstXI and BstE2 and ligation into BstXI- and BstE2-digested pSP65, and subcloned into pCMV5, as described above. K 299 M-Fyn and R 176 K-Fyn constructs were generated using the Quickchange site-directed mutagenesis kit (Stratagene Inc.). Fyn(16)-GFP was generated by PCR, using a sense oligonucleotide, encoding an EcoRI site followed by the first 16 amino acids of human Fyn fused in frame to amino acid 2 of eGFP, and an antisense oligonucleotide corresponding to the carboxy terminus of eGFP and an XbaI site. These primers were used with pEGFP-N1 as a template to generate a 1.6-kb DNA fragment that was digested with EcoRI and XbaI, followed by ligation into pCMV5. COS-1 cells (American Type Culture Collection) were cultured and transfected as previously described ( 41 ). For cotransfection experiments with Fyn and NMT, 2 μg Fyn cDNA was used with 4 or 10 μg NMT cDNA (ratios 1:2 and 1:5). For CD8-ζ coimmunoprecipitation experiments, 5 μg pEFBOS-CD8-ζ was used with 5 μg pCMV5-Fyn construct and 10 μg pCMV5-NMT or pCMV5. Transfected COS-1 cells were starved for 1 h at 37°C in DMEM minus methionine/cysteine, supplemented with 2% dialyzed FBS, and labeled for 4 h at 37°C with 25 μCi/ml Tran 35 S-label. For radiolabeling with fatty acid analogues, cells were starved for 1 h at 37°C in DMEM containing 2% dialyzed FBS, followed by radiolabeling for 4 h at 37°C with either 25 μCi/ml 125 I-IC13 or 125 I-IC16 ( 1 , 41 ). Fractionation into cytosolic (S100) and total membrane (P100) fractions and extraction with the nonionic detergent Triton X-100 was performed as described previously ( 41 ). 2-Hydroxymyristate was stored as a 100-mM stock solution in DMSO and used for experiments at 1 mM in DMEM containing 1% defatted BSA. Before addition to cells, 1 mM 2-hydroxymyristate solution was sonicated briefly and filtered to remove any undissolved myristate analogue. After preincubation for 2 h at 37°C, DMEM containing 5% FBS was added, followed by overnight incubation at 500 μM 2-hydroxymyristate. Where indicated, cells were pretreated for 1 h at 37°C with 10 μg/ml filipin in DMEM containing 1% defatted BSA. For immunoprecipitation, each clarified lysate in RIPA buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate) was mixed with 3 μl anti–Fyn or 3 μl anti–GFP and 10 μl protein A–agarose solution, and incubated for 2–12 h at 4°C. Immunoprecipitates were washed twice with RIPA buffer, once with STE (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA), resuspended in SDS sample buffer containing 0.1 M DTT, and applied to SDS-PAGE. Radiolabeled proteins in dried gels were visualized by exposure for 8–24 h to X-Omat or BioMax film ( Eastman Kodak Co. ) or to PhosphorImager screens. Quantitation of radiolabeled proteins was performed using the ImageQuant software provided with the PhosphorImager system (Molecular Dynamics, Inc.). In each labeling experiment, constructs were labeled in duplicate with 125 I-IC13, 125 I-IC16, or Tran 35 S-label, and the mean of the duplicates was calculated and used as the value for each individual experiment. Numbers were corrected for the background in each individual lane of the gel. Efficiency of myristoylation was calculated as 125 I-IC13 incorporation per unit of 35 S-label, which is accurate since all Fyn constructs used in this report (see Table I ) contain identical amounts of methionines and cysteines. Numbers for myristoylation of mutant constructs were expressed relative to wild-type Fyn within each experiment. Each construct was analyzed in two to four experiments. 10% wt/vol stock solutions of high purity digitonin ( Calbiochem Corp. ) were prepared according to the manufacturer's instructions. After prolonged storage at 4°C, stocks produce precipitates that were removed by reheating and filtration. For experiments, digitonin lysis buffer (1% digitonin, 25 mM Tris, pH 8.0, 150 mM NaCl, 300 mM KCl, 25 mM NaF, 0.1 mM Na 3 VO 4 ) was prepared freshly using a clear 10% digitonin stock. Transfected COS-1 cells were lysed for 15 min at 4°C in digitonin buffer supplemented with protease inhibitors (1.5 μg/ml of each leupeptin, aprotinin, and pepstatin A [ Boehringer Mannheim ], 20 μg/ml each of AEBSF, TLCK, TPCK, and benzamidine [ Calbiochem Corp. ]). Where indicated, the digitonin lysis buffer was supplemented with 60 mM n -octylglucoside. Lysates were centrifuged for 15 min at top speed in an Eppendorf microfuge at 4°C and incubated for 4 h at 4°C with 2 μl anti–CD8 antibody solution and 10 μl protein A/G + agarose, or with 10 μl protein A/G + agarose alone. The anti–CD8 immunoprecipitates were centrifuged for 2 min at top speed in an Eppendorf centrifuge, washed twice with digitonin buffer, and once with ST buffer (10 mM Tris, pH 7.4, 150 mM NaCl). Western blotting analysis for Fyn was performed as described ( 43 ), using a rabbit anti–Fyn polyclonal in the primary antibody step. To evaluate the levels of CD8-ζ expression, Fyn blots were stripped and reprobed with monoclonal anti–CD8 solution. As controls for total Fyn expression, supernatants of the anti–CD8 immunoprecipitation step were supplemented with 5× RIPA buffer, 3 μl rabbit anti–Fyn antibody, 10 μl protein A–agarose and incubated for >4 h at 4°C. Immunoprecipitates were washed three times in RIPA buffer and subjected to Western blotting using monoclonal antibody against Fyn (Transduction Laboratories) ( 43 ). The first set of experiments was designed to quantitatively assess the contributions of lysine residues within the Fyn SH4 motif towards fatty acylation. COS-1 cells were transfected with cDNAs encoding either wild-type Fyn, G 2 A-Fyn, or the K 7,9 A-Fyn construct, in which lysines at positions 7 and 9 are replaced by alanine residues (Table I ). Radiolabeling with Tran 35 S-label, followed by immunoprecipitation of Fyn, SDS-PAGE and autoradiography, showed that all constructs were expressed at similar levels . The K 7,9 A-Fyn construct displayed moderately elevated levels of expression and migrated as a doublet . Wild-type Fyn was labeled with both 125 I-IC13 (a myristate analogue) and 125 I-IC16 (a palmitate analogue), while G 2 A-Fyn was not , as described previously ( 1 , 41 , 43 ). Although the K 7,9 A-Fyn construct contains both the glycine at position 2 and the cysteine at position 3 required for efficient myristoylation and palmitoylation of Fyn ( 1 , 41 , 43 ), levels of 125 I-IC13 and 125 I-IC16 incorporation were drastically reduced . In subsequent experiments, K 7 A-Fyn and K 9 A-Fyn mutants, containing alanine replacements of either lysine 7 or 9, were included to analyze the relevance of the individual lysines for fatty acylation. Radiolabeling with Tran 35 S-label and 125 I-IC13, followed by calculation of expression levels and relative myristoylation (Table II ), showed that mutation of lysine 7 or 9 individually reduced myristoylation of Fyn by 51 and 40%, respectively (Table II ). Myristoylation of the double mutant, K 7,9 A-Fyn, was inhibited by 83% (Table II ), implying that the effect of each lysine mutation on myristoylation is additive. In agreement with the dependence of palmitoylation on myristoylation, the level of 125 I-IC16-palmitate labeling of K 7,9 A-Fyn was also found to be reduced to 22 ± 3% ( n = 4) of wild-type levels. Lck is the only Src family member that lacks lysines at positions 7 and 9 ( 36 , 37 ). Quantitative analysis of fatty acylation of full length Lck was hampered by inconsistent immunoprecipitation results using different batches of anti–Lck antibody. Therefore, to test whether the absence of lysines 7 and 9 impairs myristoylation of the Lck amino terminus, we used an Lck(10)Fyn fusion construct (Table I ). As shown in Table II , myristoylation of Lck(10)Fyn was very similar (∼90%) to that of Fyn, and palmitoylation of Lck(10)Fyn was ∼1.4× more efficient than that of Fyn (data not shown). These results show that despite the lack of lysines 7 and 9 in Lck, the overall fatty acylation profile at its amino terminus is very similar to that of Fyn. It is relevant to note that Lck contains a serine at position 6 (Table II ), a preferred residue at this site for mammalian NMT recognition ( 17 ). Since fatty acylation of Fyn is essential for membrane binding ( 1 , 41 , 43 ), the reduced levels of fatty acid modification of K 7,9 A-Fyn would be expected to alter the subcellular distribution of this construct. Transfected cells expressing the various Fyn constructs were radiolabeled with Tran 35 S-label, 125 I-IC13 or 125 I-IC16, followed by homogenization and fractionation into high-speed supernatant (S100) and membrane (P100) fractions, and the distribution of Fyn was analyzed. Fig. 2 shows that more than 95% of wild-type Fyn was recovered from the membrane fraction as dually acylated protein, whereas ∼75% of G 2 A-Fyn was recovered from the soluble fraction, in agreement with previous results ( 1 , 41 , 43 ). In addition, slightly more than 50% of K 7,9 A-Fyn was found in the soluble fraction , specifically consisting of the protein band with the slower migration, that is neither myristoylated nor palmitoylated (A). The reason for this difference in migration is not clear, and it cannot be explained simply by the absence of fatty acylation since the G 2 A-Fyn mutant does not show this effect. The remainder of K 7,9 A-Fyn was recovered from the membrane fraction as dually acylated protein . The calculated ratio of 125 I-IC13 label to Tran 35 S-labeled in the P100 fraction was 25% lower for K 7,9 A-Fyn compared with wild-type Fyn (data not shown), implying that some of the protein in the membrane fraction is not acylated. This is confirmed by the results with G 2 A-Fyn, for which ∼25% of the protein is found in the membrane pellet, even though none of the protein is fatty acylated . Cotransfection experiments were performed to investigate whether the reduced myristoylation of the K 7,9 A-Fyn mutant was related to levels of NMT activity inside cells. COS-1 cells were transfected with Fyn or K 7,9 A-Fyn cDNA alone , or cotransfected with cDNA encoding either wild-type NMT (lanes 3 and 4) or H 218 N-NMT (lanes 5 and 6). H 218 N-NMT contains a point mutation in the active site that abolishes transfer of myristoyl-CoA to protein ( 28 ), and serves as a negative control for the requirement of NMT activity. The ratio of NMT cDNA to Fyn cDNA used for transfection was 2:1. Fig. 3 shows that Fyn (lanes 1, 3, and 5) and K 7,9 A-Fyn protein (lanes 2, 4, and 6) were expressed at similar levels, with K 7,9 A-Fyn exhibiting an extra band with a slower migration and reduced myristoylation relative to wild-type Fyn (compare lanes 1 and 2). Cotransfection of K 7,9 A-Fyn with wild-type NMT increased 125 I-IC13 labeling two- to threefold , whereas cotransfection with enzymatically inactive H 218 N-NMT had no effect . Cotransfection of K 7,9 A-Fyn with wild-type NMT also increased 125 I-IC16 incorporation of K 7,9 A-Fyn twofold (data not shown). In addition, the relative intensity of the lower band in the K 7,9 A-Fyn doublet increased , consistent with an increase in fatty acylation levels. The data in Figs. 1 – 3 imply that levels of endogenous NMT in COS-1 cells are limiting for myristoylation of K 7,9 A-Fyn. To verify this supposition, the effect of varying the ratio of NMT cDNA to Fyn cDNA was tested. Myristoylation levels of K 7,9 A-Fyn increased twofold, using twice the amount of NMT cDNA and three- to fivefold using five times more NMT cDNA . No significant effect on myristoylation of wild-type Fyn was observed , implying that the amount of endogenous NMT was sufficient to achieve maximal myristoylation levels of this protein. Analysis of NMT expression by Western blotting confirmed that increasing levels of NMT protein were expressed after cotransfection with NMT cDNA (data not shown). Radiolabeling with Tran 35 S-label, followed by subcellular fractionation, showed that the distribution of wild-type Fyn between the soluble and the membrane fraction was not affected during coexpression of NMT . In contrast, K 7,9 A-Fyn expressed in the presence of NMT displayed a significant shift from the soluble to the membrane fraction . Taken together, these experiments demonstrate that the fatty acylation defect of the K 7,9 A-Fyn mutant can be restored by expressing excess amounts of exogenous NMT. The ability to manipulate fatty acylation of Fyn provided us with a unique opportunity to quantitate the individual contributions of the fatty acids and the surrounding amino acids towards interactions of Fyn with other membrane bound proteins. We chose to evaluate the interactions of Fyn with the ζ chain of the TCR complex as a model system. COS-1 cells were cotransfected with cDNAs for Fyn and CD8-ζ, a chimeric construct containing the extracellular and transmembrane regions of CD8, fused to the cytoplasmic tail of the TCR ζ chain ( 16 ). Cells were lysed in a 1% digitonin lysis buffer ( 10 , 33 ) and subjected to immunoprecipitation with anti–CD8 monoclonal antibody. Incubation with protein A/G + agarose beads alone was used as a negative control. When anti–CD8 immunoprecipitates were analyzed by Western blotting using polyclonal anti– Fyn antibody, a signal for Fyn was obtained using lysates from cells coexpressing Fyn and CD8-ζ . Relative to the amount of wild-type Fyn, only marginal amounts (<5%) of G 2 A-Fyn and C 3,6 S-Fyn were detected after immunoprecipitation with anti–CD8 antiserum . Since G 2 A-Fyn is neither myristoylated nor palmitoylated and C 3,6 S-Fyn is myristoylated but not palmitoylated ( 1 , 43 ), these data indicate that fatty acylation of Fyn with both myristate and palmitate is required for association of Fyn with CD8-ζ. K 7,9 A-Fyn was also impaired (∼70%) in its ability to coimmunoprecipitate with CD8-ζ , a result similar to that obtained by Gauen et al. ( 11 ) for association of K 7,9 A-Fyn with a VSV G-ζ chimera. However, the level of coimmunoprecipitation of K 7,9 A-Fyn with CD8-ζ could be increased twofold by overexpression of exogenous NMT and this correlates well with the twofold increase in myristoylation levels . This gain of function experiment indicates that the presence of lysine residues at positions 7 and 9 in Fyn is not required per se for an interaction with the ζ chain, but rather for efficient fatty acylation of Fyn. In addition, Gα o -Fyn ( 41 ) and Lck(10)Fyn, fusion proteins in which the first 10 amino acids of Fyn are replaced with those of human Gα o or human Lck (Table I ) were analyzed. Both constructs are efficiently dually fatty acylated (see reference 41 and Table II ), yet their NH 2 -terminal sequences are distinct from that of Fyn outside the met-gly-cys motif (Table I ). As depicted in Fig. 5 B, the levels of Gα o -Fyn and Lck(10)Fyn coimmunoprecipitating with CD8-ζ were similar (∼90%) to that of wild-type Fyn , providing two additional examples of the need for dual fatty acylation rather than specific lysine residues for interaction of Fyn with CD8-ζ. Controls indicated that expression levels of CD8-ζ and Fyn constructs were constant throughout the experiments . In an attempt to manipulate fatty acylation levels of wild-type Fyn, the effect of 2-hydroxymyristate, a potent inhibitor of NMT ( 25 , 27 ) was tested. Transfected COS-1 cells expressing wild-type Fyn were treated with 2-hydroxymyristate and radiolabeled with either Tran 35 S-label or 125 I-IC13. Analysis of immunoprecipitated Fyn after SDS-PAGE showed that the level of 35 S-labeled protein was unaffected but that the level of 125 I-IC13 labeling was significantly decreased (83%) after treatment with 2-hydroxymyristate . Subsequently, analysis of the subcellular distribution of Fyn after treatment with 2-hydroxymyristate showed a dramatic increase in the amount of soluble Fyn. More than 95% of Fyn was recovered from the membrane P100 fraction of control cells, whereas >40% was observed in the soluble fraction after 2-hydroxymyristate treatment . Next, the effect on coimmunoprecipitation of wild-type Fyn with CD8-ζ was analyzed. 2-Hydroxymyristate treatment caused a significant decrease in the amount of wild-type Fyn coimmunoprecipitating with CD8-ζ , to <40% of control levels. After 2-hydroxymyristate treatment, wild-type Fyn still retains glycine 2, cysteine 3, and lysines 7 and 9, all of the residues previously implicated as part of an ITAM binding motif on Fyn, yet it cannot associate with ζ. To further define the nature of the observed association between Fyn and TCR ζ, the following series of experiments was performed. Fig. 7 A shows that between 10 and 15% of the total population of Fyn coimmunoprecipitated with CD8-ζ (compare lanes 7 and 11). Fyn protein was not detected in the absence of anti–CD8 antibody during immunoprecipitation , showing that nonspecific binding of Fyn to the protein A/G + agarose beads did not occur. Fyn was only detected in anti–CD8 immunoprecipitates from cells expressing both CD8-ζ and Fyn , but not from mock transfected cells , cells expressing CD8-ζ alone , or Fyn alone . In addition, the mixing of digitonin lysates of cells that were transfected separately with either Fyn or CD8-ζ, followed by immunoprecipitation with anti–CD8-ζ antibodies, did not result in coimmunoprecipitation of Fyn , highlighting that Fyn and CD8-ζ need to be present within the same cell. Next, the functional requirements within the Fyn protein structure for association with CD8-ζ were evaluated. We first tested whether the Fyn NH 2 terminus alone is sufficient for interaction with the ζ chain in cells, using Fyn(16)GFP, a chimeric construct containing the first 16 amino acids of Fyn fused to GFP. Fyn(16)GFP expressed in COS-1 cells was dually acylated with myristate and palmitate (data not shown), and after extraction with nonionic detergent buffer, ∼60% of total Fyn(16)GFP was recovered from the detergent insoluble fraction . Thus, Fyn(16)GFP is processed and targeted like wild-type Fyn and other chimeras containing the Fyn SH4 motif ( 41 , 43 ). After coexpression of Fyn(16)GFP and CD8-ζ in COS cells, followed by digitonin lysis, no Fyn(16)GFP was detected in anti–CD8 immunoprecipitates, although there was a strong signal after immunoprecipitation with anti– GFP antibody . Similar results were obtained with constructs containing the NH 2 terminus of Fyn fused to GST or β galactosidase (data not shown). These results indicate that the NH 2 -terminal SH4 motif of Fyn by itself is not sufficient for association with CD8-ζ and that additional domains of the Fyn protein are required. To identify these requirements, CD8-ζ was coexpressed with three different Fyn mutants: K 299 M-Fyn carries a point mutation in the SH1 domain that renders Fyn kinase-inactive, ΔSH2-Fyn lacks amino acids 144–248 encoding the Fyn SH2 domain, and R 176 K-Fyn contains a point mutation of a critical arginine residue in the SH2 domains of Src family kinases that reduces phosphotyrosine binding ( 26 ). Less than 15% of ΔSH2-Fyn and only ∼35% of K 299 M-Fyn and R 176 K-Fyn was detected in anti–CD8 immunoprecipitates . The expression levels were similar for all constructs . These findings establish that functional SH1 and SH2 domains in Fyn are important for optimal interaction with CD8-ζ. Based on the findings described above that kinase activity and the SH2 domain in Fyn are critical, we addressed the relevance of the ITAM motifs in the ζ chain for association with Fyn. First, the tyrosine phosphorylation status of CD8-ζ was tested by immunoprecipitation with anti–CD8 antibody followed by antiphosphotyrosine Western blotting analysis. As shown in Fig. 8 A, CD8-ζ was strongly tyrosine phosphorylated when coexpressed with Fyn, ΔSH2-Fyn, or R 176 K-Fyn, but not when expressed alone or when coexpressed with kinase dead K 299 M-Fyn. Levels of CD8-ζ protein were constant . As observed elsewhere ( 18 ), CD8-ζ is present in three forms in COS-1 cells, migrating at 36, 38, and 41 kD, respectively . Comparison of the migration profiles indicated that the 41-kD band and the smear above it represent the tyrosine phosphorylated forms of CD8-ζ . Next, the specificity of ζ chain ITAM phosphorylation by Fyn was analyzed by use of a CD8-ζ construct in which the cytoplasmic tail was truncated after the second ITAM motif (CD8-ζT76), and a CD8-ζT76 construct in which all four remaining ITAM tyrosines were mutated to phenylalanine (CD8-ζ4F) ( 22 ). As measured by radiolabeling and immunoprecipitation, the total levels of protein expression in COS-1 cells were similar for each construct . Truncated CD8-ζT76 was observed as one main form that migrated at ∼40 kD and the major radiolabeled form of CD8-ζ4F was observed to migrate at ∼35 kD, as indicated by the arrows. The 40-kD band of CD8-ζT76 was tyrosine phosphorylated when coexpressed with Fyn, with moderately reduced levels compared with full length CD8-ζ, whereas no tyrosine phosphorylation was observed for the CD8-ζ4F mutant . Analysis of the association of Fyn with the different CD8-ζ constructs showed that similar amounts (∼90%) of Fyn coimmunoprecipitated with CD8-ζT76, whereas very little (<20%) Fyn was observed in the CD8-ζ4F immunoprecipitate . These results show that phosphorylation of ITAM tyrosines in the ζ chain by Fyn is critical for association between Fyn and ζ. A similar finding was reported by Pleiman et al. ( 29 ), who showed that the SH2 domains of Fyn and Lyn bind to the tyrosine phosphorylated ITAM in the Igα receptor. Dual fatty acylation has previously been shown to be required for localization of Fyn to plasma membrane microdomains known as rafts ( 5 , 35 , 38 , 41 , 43 ). To test for the involvement of rafts in directing the interactions between Fyn and ζ chain, two different approaches were used. First, we analyzed the sensitivity of coimmunoprecipitation of Fyn with CD8-ζ to treatment with octylglucoside and filipin, agents that disrupt raft stability ( 5 , 38 ). The association of Fyn with CD8-ζ was almost entirely abolished by addition of octylglucoside to the digitonin lysis buffer (Table III ). The interaction was also greatly reduced (∼50%) by pretreatment of cells with filipin (Table III ), an agent that binds cellular cholesterol, an essential component for raft integrity ( 5 , 38 ). An alternate approach was based on the recent observation that, in contrast to fatty acylated proteins, farnesylated proteins are largely excluded from detergent-resistant membrane rafts ( 23 ). Fyn was tagged at the carboxy terminus with the KRas4B tail (FynKRas), which promotes farnesylation and plasma membrane targeting ( 15 ). This construct is both fatty acylated and prenylated (data not shown). A second construct containing only a functional farnesylation signal was generated by mutating the amino-terminal fatty acylation sites (G 2 A,C 3 S-FynKRas). When expressed in COS-1 cells, wild-type Fyn and the two KRas-tagged constructs were completely membrane bound . Wild-type Fyn and FynKRas were also enriched in the Triton X-100 insoluble fraction . In contrast, the amount of G 2 A,C 3 S-FynKRas in the Triton X-100 insoluble fraction was significantly lower , suggesting that farnesylated Fyn exhibits reduced association with plasma membrane rafts. Fig. 9 C and Table III show that coimmunoprecipitation of G 2 A,C 3 S-FynKRas with CD8-ζ was significantly reduced, whereas association of FynKRas with CD8-ζ was very similar to that of wild-type Fyn. FynKRas and G 2 A,C 3 S-FynKRas kinase activity and ability to phosphorylate ζ ITAM tyrosines were similar to wild-type Fyn (data not shown). These observations imply that fatty acylation not only provides plasma membrane binding of Fyn, but also guides specific localization to membrane subdomains, which is essential for stabilization of the protein–protein interactions between Fyn and the ζ chain. Lysine residues at positions 7 and 9 are conserved in nearly all Src family members ( 36 , 37 ), and, in the context of the NH 2 terminus of v-Src, the lysine at position 7 is critical for efficient myristoylation ( 6 , 19 ). We were therefore interested in testing the fatty acylation status of K 7,9 A-Fyn, a mutant containing alanine substitutions for lysines 7 and 9. To obtain sensitive measurements of myristoylation levels, different Fyn constructs (Table I ) were expressed at high levels in COS cells, and radiolabeling with the iodinated myristate analogue 125 I-IC13 was performed. After immunoprecipitation and SDS-PAGE, quantitation of 125 I-IC13 incorporation into Fyn was accomplished by PhosphorImaging ( 2 ), which is not feasible after radiolabeling with conventional 3 H-labeled myristate analogues used in other studies ( 11 ). Since all Fyn constructs used in this report contain the same number of methionines and cysteines, labeling with Tran 35 S-label in parallel gave precise numbers for expression levels of the protein backbone. Subsequent calculation of 125 I-IC13 per unit of 35 S-labeled protein provided a reliable measure of the efficiency of myristoylation for wild-type and mutant Fyn constructs. Our results show that, as previously established for v-Src ( 6 , 19 ), lysine 7 is required for efficient myristoylation of Fyn (Table II ). In addition, we show that lysine 9 plays an important role in guiding effective myristoylation of Fyn (Table II ). Interestingly, the effects of the individual lysine replacements add up to the total effect observed with the double K 7,9 A-Fyn mutant , suggesting that each lysine residue plays an independent role. The conclusion that these lysines are directly involved in a myristoylation event is further substantiated by our finding that the K 7,9 A-Fyn myristoylation defect is specifically reversed by coexpression with wild-type human NMT, but not with enzymatically impaired H 218 N-NMT ( 28 ) . Reversal of the K 7,9 A-Fyn myristoylation defect by additional exogenous NMT in our experiments was almost complete . In the experiments described here, we have used cDNA encoding a 49-kD form of human NMT ( 28 ), which has full enzymatic activity in vitro and is capable of functionally complementing myristoylation deficiency in yeast ( 9 ). Additional cellular forms of NMT, with higher molecular weights (∼60 kD) and different intracellular distribution patterns, have been observed ( 14 ), and recently a second mammalian NMT was characterized ( 13 ). Most notably, missing from the 49-kD form of NMT ( 9 , 28 ) is an NH 2 -terminal sequence containing a polybasic motif that is proposed to function in ribosomal targeting of NMT ( 14 ), as well as other cotranslationally active enzymes, such as N -methionylaminopeptidases ( 21 ). We have observed that neither the 60-kD form nor the NMT 1 or 2 isoforms ( 13 ) reverse the myristoylation defect in K 7,9 A-Fyn more efficiently than the 49-kD form (data not shown). Lck is the only Src family member that lacks lysines at positions 7 and 9 ( 36 , 37 ) and one might question whether myristoylation of Lck is impaired relative to the other Src-related kinases. Since quantitation of fatty acylation of full length Lck proved technically difficult, we used an Lck(10)Fyn fusion construct (Table I ). Our results show that the fatty acylation profile of the Lck amino terminus is very similar to that of Fyn (Table II ), despite the absence of lysines 7 and 9. We infer that in Lck the serine at position 6 (Table I ), a preferred residue at this site for recognition by mammalian NMT ( 17 ) is sufficient to direct efficient fatty acylation of this sequence. If myristoylation levels of full length Lck were substoichiometric, one would expect that either its membrane association would not be complete or that additional factors (e.g., CD4) would be required to guide membrane binding of Lck. However, nearly all of the Lck in cells appears to be membrane bound ( 18 , 46 ). Moreover, two recent studies have shown that plasma membrane targeting of Lck can be achieved in the absence of CD4 and that the first 10 amino acids of Lck are sufficient for plasma membrane binding ( 4 , 46 ). These experiments imply that myristoylation and palmitoylation of full length Lck occur efficiently. Several earlier reports have described coimmunoprecipitation of the thymic isoform of Fyn with the TCR/CD3 complex ( 10 , 12 , 33 ). The association was only observed under mild detergent conditions and the exact nature of the interaction remained unclear. Recently, the first 10 amino acids of Fyn were shown to be essential for specific binding of Fyn to the ζ subunit, as well as other chains of the TCR complex in heterologous systems ( 10 ). On the basis of mutagenesis experiments, it was concluded that the amino acids glycine 2, cysteine 3, and lysines 7 and 9 define a motif for binding to ITAMs and for plasma membrane localization ( 11 ). Our results described here , as well as earlier work from our group and others ( 1 , 35 , 41 , 43 ), indicate that these amino acids within the Fyn SH4 domain are essential for optimal fatty acylation and membrane binding of Fyn. Using the coimmunoprecipitation of various Fyn constructs with a CD8-ζ fusion protein ( 16 ) as a model, we present three lines of evidence to demonstrate that the Fyn SH4 domain does not represent a specific ITAM binding motif. First, we show that the reduced coimmunoprecipitation of the K 7,9 A-Fyn mutant with CD8-ζ is specifically reversed by overexpression of NMT . The effect of NMT overexpression is to increase fatty acylation and membrane binding of K 7,9 A-Fyn . This gain of function experiment clearly establishes that coimmunoprecipitation of Fyn with CD8-ζ does not require lysines 7 and 9 per se, but their presence is crucial for directing efficient fatty acylation. This conclusion is further substantiated by the observations made with the Gα o -Fyn and Lck(10)Fyn constructs, which coimmunoprecipitate with CD8-ζ as efficiently as wild-type Fyn . The NH 2 -terminal sequences of Gα o -Fyn and Lck(10)Fyn do not contain lysines at position 7 and 9 (Table I ), but they are efficiently fatty acylated (reference 41 , and Table I ). Second, the loss of function experiment with 2-hydroxymyristate shows that, even in the presence of all the amino acids proposed to constitute the ITAM binding motif (glycine 2, cysteine 3, and lysines 7 and 9), coimmunoprecipitation of wild-type Fyn with CD8-ζ can be ablated. Thus, loss of association of wild-type Fyn to CD8-ζ coincides with a reduction of fatty acylation and membrane binding . Third, Fyn(16)GFP did not coimmunoprecipitate with CD8-ζ , showing that the first 16 amino acids of Fyn are not sufficient for interaction with the ITAMs on ζ, in agreement with the results of cross-linking studies ( 34 ). This implies that other protein determinants in Fyn are involved in mediating the interactions with ζ chain. Indeed, coimmunoprecipitation of Fyn was observed to require both a functional kinase as well as an SH2 domain . Coimmunoprecipitation of Fyn with TCR subunits occurs only under mild detergent conditions ( 10 , 12 , 33 ), which has been taken as evidence for low affinity protein–protein interactions. Our findings with Fyn, and a recently growing interest in membrane microdomains ( 5 , 38 ), suggests the involvement of additional mechanisms for stabilization of the interactions between Src kinases and TCR subunits, which is supported by several recent independent reports ( 24 , 44 , 45 ). In biological membranes, especially plasma membranes, phase-separated “rafts” exist, representing lipid domains enriched in cholesterol and sphingolipids that provide scaffolds for clustering of transmembrane and peripherally associated proteins ( 5 , 38 ). These rafts or microdomains can be recovered from cells using mild detergent conditions ( 5 ). There is growing evidence that key components of TCR-mediated signaling events are localized to rafts. For example, palmitoylation of the T cell transmembrane protein LAT is essential for localization in rafts and T cell tyrosine phosphorylation events ( 45 ). Src family kinases, especially Fyn and Lck, are constitutively localized in rafts via dual fatty acylation motifs ( 35 , 41 , 43 ). Activation of the TCR results in specific accumulation of ζ chains in plasma membrane rafts ( 24 ). In COS cells, ∼20% of CD8-ζ is in the detergent-insoluble fraction (data not shown), similar to the distribution of the ζ chain in resting T cells ( 7 ), and this may explain in part why only a limited amount (<20%) of total Fyn coimmunoprecipitates with CD8-ζ in COS cells. The functional significance of raft localization is evidenced by the finding that disruption of cell surface rafts interferes with early T cell signaling events ( 44 ). These observations establish a connection between local accumulation of signaling molecules in plasma membrane rafts and regulated T cell signaling events. Based on the results reported here and by others ( 35 , 39 , 41 , 43 ), the following model emerges. Myristoylation and palmitoylation within the NH 2 -terminal SH4 domain directs Fyn into detergent-resistant plasma membrane rafts and positions the downstream SH2 and kinase domains in close proximity to the TCR ζ chain. Phosphorylation of the ζ ITAMs by Fyn allows subsequent interaction between the phosphorylated ITAMs and the Fyn SH2 domain . Accumulation of Fyn and the ζ chain in rafts increases the local concentration of both proteins and drives their association. The stability and the extent of Fyn SH2/ζ phosphotyrosine interaction is apparently dependent on the integrity of the rafts since treatment with octylglucoside and filipin, agents that disrupt rafts ( 5 , 38 ), results in dissociation of the Fyn/ζ complex (Table III ), presumably due to a decrease of the effective local protein concentrations. This implies that the Fyn SH2/ζ phosphotyrosine interaction is relatively weak, as found for several other SH2/phosphotyrosine interactions ( 20 ). In T cells, dissociation of the Fyn SH2 domain from the ζ chain would make ITAM phosphotyrosine residues on ζ accessible for binding to the SH2 domains of other signaling molecules, such as ZAP-70. This model explains why undermyristoylated and underpalmitoylated forms of Fyn, that are not efficiently membrane bound, as well as a farnesylated Fyn construct that is not enriched in membrane subdomains , do not interact well with the ζ chain. These studies illustrate that the role of the NH 2 -terminal SH4 motif is to properly position Src kinases within specific locations of cellular membranes for optimal interaction of downstream Src homology domains with other membrane-bound signaling molecules. | Study | biomedical | en | 0.999996 |
10209032 | mAb SYN1351 recognizes all syntrophin isoforms . Syntrophin isoform-specific polyclonal antibodies SYN17 (α1-syntrophin), SYN28 (β2-syntrophin), and SYN37 (β1-syntrophin; all used at 30 nM IgG) have been described previously . mAb MANCHO-3 (a gift of G.E. Morris) is described elsewhere . Antibody 13H1 recognizes both α- and β-dystrobrevins . Antibody NCL-DYS2 (epitope in the COOH terminus of dystrophin) is known to cross-react with the canine protein (Novacastra). mAb 1808 raised against Torpedo dystrophin recognizes the rod domain of dystrophin . Monoclonal anti–α-dystroglycan antibody, VIA4-1 (Upstate Biotechnology Inc.), is described elsewhere . Monoclonal and polyclonal anti-GFP (green fluorescent protein) antibodies ( Clontech Laboratories, Inc. ), anti–β-catenin ( Santa Cruz Biotechnology ), anti–ZO-1 ( Zymed Labs, Inc. ), and anti-Na/K ATPase (Chemicon International, Inc.) were used according to manufacturers' specifications. The following mouse β2-syntrophin constructs were subcloned using common restriction sites into pEGFPC2 expression vector ( Clontech Laboratories, Inc. ): full-length (FL; aa 1–520); PH1 (aa 1–94 and 175–288); PDZ (aa 90–185); PH2 (aa 296–425); and syntrophin unique (SU; aa 421–520). α1-syntrophin PDZ (aa 75–171) and β1-syntrophin PDZ (aa 105–200) were also subcloned into the same vector. The PH2SU (aa 296–520) and PH1PDZ (aa 1–288) constructs were amplified by PCR and were subcloned using engineered restriction sites. All constructs were sequenced before use. Type II MDCK cells were transfected using lipofectamine ( GIBCO BRL ) according to manufacturer's recommendations. After 10– 14 d of growth in selection medium (DME + 5% FBS + 400 μg/ml G418), individual colonies were isolated and stable lines were established. Wild-type MDCK cells were maintained in DME + 5% FBS. Cells were grown to confluence on glass coverslips (5–7 d), fixed in 2% paraformaldehyde for 15 min, and either analyzed for GFP expression immediately, or prepared further for immunofluorescence. Cells were permeabilized for 15 min in PBS/0.5% Triton X-100, blocked in PBS/1% fish gelatin/0.8% BSA for 30 min, and incubated with primary antibodies at the appropriate dilution for 1 h at room temperature or overnight at 4°C. After washing in PBS/0.5% Triton X-100, cells were incubated with Alexa 488–conjugated secondary antibody (Molecular Probes, Inc.) and either Texas red–conjugated (Jackson ImmunoResearch Laboratories, Inc.) or Alexa 594–conjugated (Molecular Probes, Inc.) secondary antibody for 1 h at room temperature. Cells were washed with PBS/0.5% Triton X-100 and mounted onto slides in glycerol with n -propyl gallate to minimize fading . Staining was analyzed using confocal microscopy (Leica TCS-NT). Results were similar when MDCK cells were grown for 5–7 d on transwell filters (Corning Costar). Wild-type or GFP-expressing MDCK cell lines were grown on 100-mm plates for 5–7 d. Cells were rinsed in ice-cold homogenization buffer (HB; 10 mM sodium phosphate, 0.4 M NaCl, 5 mM EDTA, pH 7.8) and lysed for 30 min on ice in HB/1% Triton X-100 (750 μl/plate) with protease inhibitors (2 mM PMSF, 1 μM bestatin, and 1 μg/ml each of aprotinin, leupeptin, antipain, and pepstatin A). Insoluble proteins were pelleted at 39,000 g for 30 min. The soluble extracts were then incubated with 1 μg of specific antibody or control IgG for 1 h at 4°C. Protein A– or G–agarose ( Sigma Chemical Co. ; 25 μl of 50% slurry) was added and incubated overnight at 4°C. Beads were washed extensively in HB containing 1 M NaCl/ 1% Triton X-100. Proteins eluted in SDS sample buffer were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting as described previously . In brief, after blocking in TBS/ 0.1% Tween 20 (TBST) with 5% milk for 1 h at room temperature, nitrocellulose was incubated with primary antibody diluted in TBST/1% milk for 1 h at room temperature. Blots were washed 3 times for 15 min each in TBST, and were then incubated with an HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Blots were washed as before and signal was detected using enhanced chemiluminescence ( Pierce Chemical Co. ) and exposed to film. Nitrocellulose blots were occasionally reprobed after stripping with stripping buffer (Chemicon International, Inc.). Syntrophins are found in many tissues in which epithelia are a major cell type, including lung and kidney . However, these tissues contain a mixture of epithelial and nonepithelial cells. To study the expression and distribution of syntrophins in polarized epithelial cells, we used MDCK cells. Expression of components of the DAPC was determined by immunoblotting of Triton X-100 soluble and insoluble fractions from MDCK cells. An mAb that recognizes all three syntrophin isoforms detected a single band of 60 kD . Most of this protein was found in the Triton X-100 soluble fraction. Utrophin, a dystrophin homologue, was also found and, like syntrophins, was detected primarily in the Triton X-100 soluble fraction. Syntrophin and utrophin were also found in two additional polarized epithelial cell lines: HBE, a human bronchial epithelia cell line; and LLCPK, a pig renal epithelia cell line (data not shown). The expression of syntrophin isoforms differs among tissue types. For instance, skeletal muscle contains all three syntrophin isoforms, while β1-syntrophin is the predominant form in liver . To identify the syntrophin isoforms expressed in polarized epithelia, samples enriched for syntrophins by immunoprecipitation with SYN1351 were analyzed by immunoblotting with antibodies specific for each of the three syntrophin isoforms. β2-Syntrophin was the only syntrophin isoform consistently detected , although in some experiments small amounts of α1-syntrophin were also found. Given its abundance in the epithelial cell lines tested and in tissues rich in epithelial cells, β2-syntrophin appears to be the predominant form of syntrophin in epithelia. In skeletal muscle, syntrophins associate directly with dystrophin family members . This also appears to be the case in MDCK cells. Utrophin and a form of dystrobrevin were specifically enriched in SYN1351 preparations, but were absent from control preparations . These results indicate that syntrophin, utrophin, and dystrobrevin exist in a stable complex in polarized epithelial cells. Polarized epithelial cells have two distinct membrane domains that differ greatly in lipid and protein content . To determine the localization of the syntrophin/ utrophin complex, we compared the distribution of syntrophin with ZO-1, β-catenin, and Na/K ATPase, markers of distinct membrane specializations in polarized MDCK cells. The distribution of syntrophin was very similar to that of Na/K ATPase, which is largely basolateral. Syntrophin outlined the cell on its basal and lateral surfaces, but was absent from the apical membrane. The lateral membrane of polarized epithelial cells contains two specialized structures, the TJ and the adherens junction. The TJ is located at the intersection of the apical and basolateral membrane, where it acts to regulate diffusion across the epithelium and as a barrier between the apical and basolateral membrane . The adherens junction is a cadherin-based specialization believed to be involved in adhesion of epithelial cells . In addition to providing useful markers for subdomains within the lateral membrane, the TJ and adherens junction are key structures of epithelial cells where numerous PDZ-containing proteins have been shown to be important . Therefore, we were interested in the relationship between the syntrophin complex and these junctions. β-Catenin is found enriched at the adherens junction and in a cytosolic pool . As shown in Fig. 3 B, syntrophin and β-catenin are colocalized on the lateral membrane. Anti– ZO-1 specifically labeled TJs , and appeared to represent the upper limit of syntrophin expression . The distribution of utrophin was indistinguishable from that of syntrophin, having the same limits of expression when double labeled with anti–ZO-1 antibodies . While PDZ-containing proteins have been shown to be important at synapses and cell–cell junctions , only the targeting of Dlg/SAP97 and ZO-1 has been studied extensively. As a first step in determining which domains of syntrophin are responsible for its localization on the basolateral membrane, we studied the distribution of exogenously expressed GFP fusion proteins of syntrophin . MDCK cells were transfected with a plasmid encoding GFP fused to the NH 2 terminus of full-length β2-syntrophin (GFP-FL) and multiple stable cell lines were established. GFP-FL was found exclusively on the basolateral membrane, in a distribution indistinguishable from that of endogenous syntrophin . In contrast, GFP alone was found only in the cytoplasm. Syntrophins contain four distinct domains: two PH domains, a PDZ domain, and an SU domain . Any of these domains could be involved in targeting to the basolateral membrane. Since PH domains in other proteins have been shown to be capable of binding phospholipids , the PH domain(s) of syntrophin may recruit syntrophin to the basolateral membrane through binding to specific types of lipids. This model is especially attractive since the apical and basolateral membranes differ in lipid content . Alternatively, the PDZ domain could direct syntrophin to the basolateral surface by binding a ligand that is restricted to the basolateral membrane. This could be a general mechanism by which PDZ-containing proteins are recruited to specialized sites. Finally, the SU domain, which is unrelated to any known protein domain, may be responsible for targeting syntrophin to specific subcellular compartments. To examine these possibilities, we established stable MDCK cell lines expressing individual β2-syntrophin domains fused with GFP and compared the distribution of the GFP fusion proteins with endogenous syntrophin. The PH1, PDZ, PH2, and SU domains of β2-syntrophin all failed to accumulate at the basolateral membrane . The PDZ domains of α1-syntrophin and β1-syntrophin also failed to target (data not shown). Each individual domain was distributed diffusely throughout the cell and was identical to the distribution of GFP alone . In the PH1 and PH2 cell lines, labeled cells were very sparse. We counted >200 positive cells in three independent cell lines for each construct. In all cases, the GFP-tagged domain was diffusely distributed throughout the cell. The failure of individual domains to localize preferentially to the basolateral membrane may be due to their inability to bind a partner, perhaps utrophin, which resides on this membrane. The COOH-terminal 37 kD of β1-syntrophin, consisting of part of the PH1 domain, along with the PH2 and SU domains, is sufficient to bind to dystrophin family members in vitro . Individual domains of syntrophin may not retain their ability to bind utrophin, and therefore cannot be recruited to or retained at the basolateral membrane. To test this hypothesis, we made two additional GFP constructs which consisted of tandemly linked PH2 and SU domains (PH2SU), or the linked PH1 and PDZ domains of β2-syntrophin . In multiple stable cell lines, GFP-PH2SU was found on the basolateral membrane , in a distribution indistinguishable from endogenous syntrophin or expressed GFP-FL. In contrast, GFP-PH1PDZ failed to accumulate at the basolateral membrane , indicating that the PH2SU construct is necessary and sufficient for basolateral sorting. Recently, the targeting of some GFP-tagged proteins was shown to be substratum dependent . However, we obtained similar syntrophin domain targeting results whether transfected MDCK cells were plated on glass coverslips or on transwell filters (data not shown). To test biochemically whether GFP-PH2SU retains the ability to bind utrophin while individual syntrophin domains and PH1PDZ do not, we immunoprecipitated each GFP fusion protein and determined whether utrophin was specifically coisolated. Detergent extracts from cell lines expressing GFP, or fusion proteins of full-length β2-syntrophin, PH2SU, PH1PDZ, or individual syntrophin domains were subjected to immunoprecipitation with anti-GFP or control antibody. To confirm the size and expression level of each fusion protein, immunoprecipitates were analyzed by immunoblotting with a monoclonal anti-GFP antibody. Sample loadings were then adjusted to obtain comparable amounts of each fusion protein . The top halves of the same blots were incubated with a monoclonal antiutrophin antibody. As expected, utrophin was specifically coimmunoprecipitated with GFP-FL, indicating that fusion with GFP did not interfere with the interaction between syntrophin and utrophin . Utrophin also coimmunoprecipitated with the PH2SU tandemly linked domains, but not with PH1PDZ or individual syntrophin domains . This ability of the GFP-PH2SU fusion protein to bind utrophin may underlie its localization on the basolateral membrane. A current model of the stoichiometry of the DAPC in muscle predicts one utrophin, one dystrobrevin, and two syntrophins per complex: one syntrophin binds directly to utrophin or dystrophin and another binds to a dystrobrevin family member . To test whether two syntrophins are present in epithelial syntrophin/utrophin complexes, we determined whether endogenous syntrophin copurified with exogenously expressed GFP-syntrophin . We used an antibody directed against the syntrophin PDZ domain to detect endogenous syntrophin in samples immunoprecipitated with GFP antibodies. Although SYN1351 also detects GFP-FL, GFP-PH1PDZ, and GFP-PDZ constructs , they are well separated from the endogenous syntrophin on our immunoblots (except in the case of the PH1PDZ construct, which is the same size as endogenous syntrophin). In these immunoprecipitation experiments we find that endogenous syntrophin copurifies with GFP-FL and GFP-PH2SU, but not with GFP-PH1, GFP-PDZ, GFP-PH2, or GFP-SU fusion proteins. Although we cannot determine whether endogenous syntrophin copurifies with GFP-PH1PDZ, we believe it does not since it fails to copurify utrophin and dystrobrevin. To accommodate two syntrophin binding sites, a dystrobrevin must also be present in the complex. When blots were probed with a panspecific dystrobrevin antibody, we observed a 65-kD dystrobrevin isoform in samples immunoprecipitated from GFP-FL and GFP-PH2SU cell lines . Dystrobrevin was not detected when GFP antibody was used to immunopurify individual GFP-tagged syntrophin domains or the PH1PDZ fusion protein. The failure of the PDZ fusion proteins to associate with endogenous syntrophin suggests that PDZ homodimerization does not occur with syntrophins as it does with other PDZ proteins . In this study, we have characterized an epithelial utrophin-associated protein complex expressed in MDCK cells. Our data demonstrate that this complex is restricted to the basolateral cell surface of epithelial cells, and includes β2-syntrophin and an isoform of dystrobrevin. We find that utrophin and dystrobrevin copurify with syntrophin, providing strong evidence that these proteins are associated in MDCK cells as they are in other tissues . The dystrobrevins are divided into two families, α and β, which undergo extensive alternative splicing . The molecular mass of dystrobrevin found in MDCK cells is ∼65 kD and, thus, could correspond to either α-dystrobrevin-2 or β-dystrobrevin. Although our isoform-specific antibodies are not reactive with the canine proteins, therefore preventing a direct test of this question, we believe that this MDCK protein is likely β-dystrobrevin for two reasons. First, β-dystrobrevin is enriched in epithelial tissues , while α-dystrobrevin-2 is restricted to brain, skeletal, and cardiac muscle . In addition, α-dystrobrevin-2 associates preferentially with dystrophin , and therefore is unlikely to be found in a utrophin complex. Taken together, these data suggest that β-dystrobrevin is the isoform present in MDCK cells. While multiple syntrophins are often expressed in the same tissue, a single isoform often predominates. For instance, α1-syntrophin is the major form in skeletal and cardiac muscle, β1-syntrophin in liver and smooth muscle, and β2-syntrophin in epithelial-rich tissues like kidney and lung . In tissues where β2-syntrophin is not the dominant form, such as brain and muscle, it is often restricted to sites of membrane specialization, such as the neuromuscular junction and retinal synapses (Peters, M.F., C. Houlihan, and S.C. Froehner, unpublished results). Thus, β2-syntrophin may play a unique role at membrane specializations. Here, we find that β2-syntrophin is the dominant syntrophin isoform expressed in MDCK cells. The predominance of β2-syntrophin could be explained by the failure of the α1-syntrophin and two β1-syntrophin isoform specific antibodies to recognize canine syntrophins. However, the abundance of β2-syntrophin in epithelial-rich rodent tissues and in a human bronchial epithelial cell line (data not shown), in which our antibodies are known to be reactive, suggests that β2-syntrophin is the major isoform in epithelia. The syntrophin/utrophin complex of MDCK cells probably includes the dystroglycans. We find α-dystroglycan on the basolateral membrane of MDCK cells (data not shown) consistent with report of dystroglycans on the basal surface of epithelial cells in vivo . These data support a model in which the syntrophin/utrophin complex serves as a link between the ECM and the actin cytoskeleton in epithelia, as it does in skeletal muscle. There have been reports of a dystrophin short form, Dp140, on the basal surface of tubule epithelial cells in kidney . While we did detect some dystrophin by immunofluorescence in MDCK cells (data not shown), it was not found at the basolateral membrane. Thus, dystrophin may be important in certain epithelia, but we do not believe it is part of the basolateral complex in MDCK cells. The region on dystrophin that binds syntrophins has been mapped to a short segment encoded by exon 74 . However, identification of the region of syntrophin responsible for binding to dystrophin family members has not been reported. Binding studies using in vitro translated proteins showed that a 37-kD fragment containing the COOH terminus of β1-syntrophin is sufficient for association with dystrophin family members . We find that individual PH2 or SU domains fail to bind utrophin, but when these domains are tandemly linked (as they are in the native protein) binding to utrophin is restored. These data suggest that the combined PH2 and SU domains function as a unit. Perhaps the binding site for utrophin is formed by noncontiguous partial binding sites in the PH2 and SU domains. Alternatively, the region of syntrophin responsible for binding utrophin may bridge sequences in the PH2 and SU domains. Although the individual constructs used for expression of PH2 and SU domains overlapped by four amino acids, it is possible that proper folding of this bridge region requires a longer polypeptide. Finally, it is possible that the utrophin binding site is contained within a single domain, but that both domains are required for proper folding of the interacting site. While our data do not discriminate between these possibilities, they do provide in vivo evidence that an intact PH2SU domain is necessary and sufficient for syntrophin binding to utrophin. In muscle, the current model of the DAPC includes dystrophin or utrophin directly associated with a dystrobrevin family member through a coiled-coil interaction . The presence of syntrophin binding sites in both dystrophin or utrophin, and dystrobrevin allows for the binding of two syntrophins per complex . Support for this model comes from the demonstration that dystrophin or utrophin complexes contain pairs of syntrophin isoforms and a stoichiometry of two syntrophins per complex . In this study, we tested the hypothesis that two syntrophins are contained within the epithelial syntrophin/ utrophin complex by expressing GFP-tagged syntrophin (GFP-FL) in MDCK cells. In experiments in which GFP-tagged syntrophin was immunoprecipitated with anti-GFP, we detected not only utrophin and dystrobrevin, but also endogenous syntrophin. A single syntrophin binding site on utrophin and dystrobrevin likely accounts for the two syntrophins in the purified complex. The amount of endogenous syntrophin is approximately half that of the GFP-tagged syntrophin, suggesting that some purified complexes contain two GFP-tagged syntrophins, while others contain one GFP-tagged syntrophin and one endogenous syntrophin. Whether GFP-tagged syntrophin displaces endogenous syntrophin or instead binds to unoccupied sites on utrophin and dystrobrevin is unclear. Alternatively, our results could also be explained by dimerization of syntrophins within the complex. Although evidence for syntrophin dimerization has been reported , alternative explanations for these findings have been suggested . Our data indicate that if syntrophin dimerization does occur, it must be via the PH2 and SU domains, since none of the other constructs were able to copurify endogenous syntrophin. Epithelial cells contain distinct apical and basolateral cell surfaces with unique protein and lipid compositions . The epithelial syntrophin/utrophin complex is restricted to the basolateral cell surface. As a first step in understanding the sorting of the syntrophin/utrophin complex to the basolateral membrane of epithelial cells, we investigated the targeting of syntrophin in MDCK cells. Our results show that the tandemly linked PH2 and SU domains are necessary and sufficient for directing GFP-tagged syntrophin to the basolateral membrane . The PH domain and additional COOH-terminal sequences are also required to target cytohesion-1 and Bruton's tyrosine kinase to the plasma membrane . However, it is not simply the presence of a PH domain which confers membrane targeting of syntrophin since the PH1PDZ construct failed to accumulate at the basolateral membrane. Interestingly, replacement of the cytohesion-1 PH domain with the PH domain of the β1-adrenergic receptor abolished membrane targeting . The amino acid residues responsible for targeting syntrophin to the basolateral membrane may also be necessary for binding to utrophin. Alternatively, syntrophin may contain two independent regions, one responsible for binding to utrophin and a second involved in binding structures at the basolateral membrane. Site-directed mutagenesis of the PH2SU domain may allow for the discrimination of utrophin-binding and basolateral targeting functions within this region of syntrophin. In epithelia, the signals responsible for polarized sorting are best characterized for transmembrane proteins. For instance, several integral membrane proteins that target to the basolateral surface in epithelial cells contain di-leucine or tyrosine-based basolateral sorting sequences . Therefore, we examined the sequence of β-dystroglycan for potential targeting sequences and found a conserved sequence, EDQATFI (amino acids 784–790 in mouse and human) in the COOH terminus of β-dystroglycan, that is very similar to the consensus sequence for the di-leucine basolateral sorting motif, DDQxxLI . In β-dystroglycan, this motif is predicted to be cytoplasmic and does not overlap with the dystrophin binding site . The cytoplasmic targeting motifs found in basolateral proteins are often followed by small clusters of acidic residues . The presence of the sequence DELDD downstream of the putative di-leucine motif in β-dystroglycan supports a role for this motif in basolateral sorting. It will be interesting to examine the importance of this sequence in the targeting of β-dystroglycan and other components of the syntrophin/utrophin complex in epithelial cells. At this time, we can only speculate as to the function of the basolateral syntrophin/utrophin complex in polarized epithelia. The syntrophin/utrophin complex, through its ability to link ECM proteins to actin, may serve a structural role in epithelia. In skeletal muscle, the link between the ECM and the cytoskeleton is thought to maintain cell membrane integrity during contraction . Some epithelia must also withstand the forces of contraction (i.e., in the gastrointestinal tract). Thus, a link between the ECM and actin provided by the syntrophin/utrophin complex, may maintain membrane integrity in epithelia. The syndecan/CASK complex also links the ECM to the actin cytoskeleton , and may play a role similar to the syntrophin/utrophin complex in epithelia. The syntrophin/utrophin complex may also function to recruit proteins to the basolateral surface of epithelial cells. Syntrophins are modular adapter proteins made up almost exclusively of protein–protein interaction domains . Our results indicate that the PH1 and PDZ domains do not play a role in targeting syntrophin to the basolateral membrane. In contrast, the PDZ domains of Dlg/SAP97 are necessary for efficient subcellular targeting , indicating that modular adapter proteins may be targeted via different mechanisms. The PH1 and PDZ domains of syntrophin are also unnecessary for the interaction of syntrophin with utrophin. Therefore, these domains are free to interact with additional proteins to generate a large multiprotein complex . Binding partners for the PH1 domain of syntrophin have not been identified, but PH domains in other proteins are capable of binding proteins such as protein kinase C, and the β and γ subunits of G proteins . The ability of the PDZ domain of syntrophin to bind nNOS in muscle suggests that one function of syntrophin is to recruit signaling proteins to the membrane. Interestingly, the presence of two syntrophins per complex may allow two different proteins to be brought in close apposition, allowing for one to modulate the other. For instance, in skeletal muscle, syntrophin PDZ domains also bind voltage-gated sodium channels . NO modulates the activity of certain sodium channels , a process that may occur with high efficiency and specificity if nNOS and sodium channels reside in the same complex. The recruitment of two proteins into the DAPC in muscle has potential functional consequences: the formation of similar complexes containing signaling molecules, and effector proteins may also occur in epithelia. Thus, it will be important to identify binding partners for the PH1 and PDZ domains to gain further understanding of the function of the syntrophin/utrophin complex in epithelia. PDZ domain-containing proteins are a common feature of many scaffolding complexes . Through their interactions with the COOH-terminal tails of receptors and ion channels, PDZ domains are critical in the assembly of multiprotein complexes. Many scaffolding proteins contain multiple PDZ domains, which may tether multiple copies of the same ligand at a particular subcellular location. More often, the PDZ domains within a single protein have distinct binding specificities, allowing different proteins to be recruited to the same subcellular location. An elegant illustration of the efficiency of such complexes comes from studies of INAD (inactivation no after potential D) where a single type of scaffold links all (or most) proteins needed for Drosophila phototransduction . Interestingly, homodimerization of INAD molecules generates a complicated network of proteins at the membrane . Furthermore, the potential for homodimerization of proteins which contain 6–13 PDZ domains , raises the complexity of scaffolding complexes to almost incomprehensible heights. In epithelia, the asymmetric localization of proteins and lipids results in functional differences between the apical and basolateral membranes required for epithelial cell function. MDCK cells sort secretory and membrane-associated proteins to apical and basolateral surfaces by several different mechanisms . Some proteins are packaged upon exit from the TGN into separate apical or basolateral transport vesicles . Other proteins are targeted exclusively to the basolateral domain but do not remain there; instead they are internalized into endosomes and targeted via the transcytotic pathway to the apical cell surface. Finally, some proteins are transported in a nonpolarized manner to both cell surfaces, but are selectively stabilized at one surface. For example, the Na/K ATPase is stabilized at the basolateral cell surface by association with the actin cytoskeleton and ankyrin . Syntrophin may play a similar role and act to specifically anchor transmembrane proteins by high affinity protein–protein interactions via the PDZ or PH domains. However, since PDZ proteins are present on both apical and basolateral cell surfaces, and at the TJs, additional factors that define binding specificities must be involved. In addition to a targeting role once a polarized monolayer is formed, the epithelial syntrophin/utrophin complex may be involved in the development of polarity in epithelial cells. Drubin and Nelson proposed that extracellular cues, such as cell–cell adhesion, define discrete areas of membrane as sites of submembranous cytoskeletal assembly. Once assembled, the cytoskeleton serves as a docking site for specific proteins, leading to further specialization of this region of membrane. In MDCK cells, E-cadherin–mediated adhesion defines the site for recruitment of the sec6/sec8 complex . This protein complex then serves as a docking site for additional proteins. By serving as a link between the ECM and the submembranous cytoskeleton, the syntrophin/utrophin complex may also assist in defining basal membranes during morphogenesis. Laminin, a component of the basement membrane which binds dystroglycan , plays an important role in the differentiation of epithelia in vivo and in vitro . Antibodies that block the binding of laminin to dystroglycan in organ cultures inhibit epithelial differentiation . Perhaps the binding of laminin serves to recruit the syntrophin/utrophin complex to the basal membrane, where it participates in the specialization of this cell surface. In addition, the heparan sulfate proteoglycan, agrin, is found in the basement membrane of epithelial tissues, where it binds dystroglycan . It is important to note that antibodies that disrupt the laminin–dystroglycan interaction may also block agrin binding , suggesting that agrin may also be involved in epithelial morphogenesis via the syntrophin/utrophin complex. The use of additional cell culture model systems and transgenic or knockout animals will help in defining functions for the epithelial syntrophin/utrophin complex. | Study | biomedical | en | 0.999997 |
10209033 | Human brain microvessels were derived from cortical tissue acquired from both temporal lobe biopsies and autopsies. Biopsies were obtained from patients undergoing surgery for the treatment of intractable seizures at the Yale-New Haven Hospital, and were resected from areas outside the epileptic foci. Autopsy tissue was acquired from the following sources: University of Miami and University of Maryland Brain and Tissue Banks for Developmental Disorders , Harvard Brain Tissue Resource Center Bank and National Disease Research Interchange (Philadelphia, PA). Final diagnoses included respiratory distress and polytrauma, with no evidence of neurologic disease. Microvessels were prepared by a modification of a previously described method for the isolation of rat brain microvessels ( 10 ). In this case, enriched fractions of microvessels obtained by centrifugation through dextran were further purified by isopycnic sedimentation through Percoll ( 4 ). This method yielded a population of microvascular segments that retained their basement membranes. Purified microvessels were reacted with either biotinylated recombinant human (rh)MCP-1 or biotinylated rhMIP-1α (R&D Systems Inc.), and subsequently with fluorescein-avidin according to the manufacturer's directions. All reactions were carried out at 4°C or room temperature to lessen possible cellular uptake of chemokines or antibodies. For negative controls, reactions were performed in the presence of anti–human MCP-1 and MIP-1α antibodies, or included the irrelevant biotinylated protein soybean trypsin inhibitor (R&D Systems Inc.). As positive controls, peripheral blood monocytes (PBM), which contain receptors for both MCP-1 and MIP-1α ( 2 , 3 , 19 , 35 ), were subjected to the identical conditions. Competition studies using a single chemokine were performed with constant concentrations of either biotinylated rhMCP-1 or rhMIP-1α in the presence of increasing concentrations of unlabeled, homologous ligand (rhMCP-1 or rhMIP-1α; both from PeproTech Inc. ). Cross-competition studies between the two chemokines were conducted using a single concentration of either biotinylated rhMCP-1 or rhMIP-1α in the presence of increasing concentrations of unlabeled, heterologous ligand. Relative amounts of biotinylated MCP-1 and MIP-1α bound to microvessels were quantified using a Zeiss LSM 410 confocal microscope equipped with an argon-krypton laser (with emission at 488 and 568 nm). Microvessels were observed with an Achromat 40×/1.3 NA, oil objective, under constant conditions of aperture, pin-hole, brightness, and contrast. Images (512 × 512 pixels) were obtained and processed using Adobe Photoshop 3.0 software (Adobe Systems Inc.). To quantify the extent of labeled chemokine binding, values of mean pixel intensity were recorded from a total of 80 randomly chosen areas (192 pixels each) of microvessels from at least 10 samples. In analogous fashion, mean pixel intensities were also obtained from microvessels incubated with biotinylated soybean trypsin inhibitor. The average of the pixel values obtained from this negative control was then subtracted from each of the 80 pixel intensities obtained from all the different chemokine binding conditions so as to remove the contribution of background noise. Corrected pixel intensities were then averaged, with the resulting value representing the relative degree of specific chemokine binding along microvessels. K d and Hill coefficient ( n ) values were determined using Sigma Plot software (Jandel Inc.) and using the following equation: b = ( b max · x n )/( K d n + x n ), where b is indicated by pixel intensity values. After reacting isolated microvessels with biotinylated chemokines and fluorescein-avidin, as described above, or for the specific detection of chemokine receptors, microvessels were fixed in 4% (wt/vol) paraformaldehyde and seeded onto poly- l -lysine–coated glass coverslips. After blocking nonspecific binding by incubation with PBS containing 5% (vol/ vol) normal goat serum ( GIBCO BRL ), 1% (wt/vol) BSA ( Sigma Chemical Co. ), and 0.5% (vol/vol) Tween ( Sigma Chemical Co. ), microvessels were reacted with the different primary antibodies (monoclonal antilaminin [clone LAM-89, Sigma Chemical Co. ], monoclonal anti–Factor VIII [clone F8/86, DAKO], monoclonal anti-CD68 [clone EBM11, DAKO], anti–collagen type IV collagen [ Sigma Chemical Co. ], and anti–heparan sulfate proteoglycan [Upstate Biotechnology]), and then exposed to rhodamine-conjugated goat anti–mouse antibody ( Boehringer Mannheim Biochemicals ). Monoclonal antibodies to chemokine receptors CCR1, CCR2, and CCR5 were all purchased from R&D Systems Inc., and samples reacted with these antibodies were subsequently visualized with fluorescein-conjugated goat anti–mouse antibody ( Boehringer Mannheim Biochemicals ). Negative control samples were processed similarly, except for the exclusion of primary antibody, while isotype control samples used similar Ig isotype, irrelevant antibody as a primary source. All samples were viewed with an Olympus IX70 inverted microscope (40× objective) to obtain simple, two-dimensional images, and a Zeiss LSM 410 confocal microscope (40×/1.3 oil objective) to acquire three-dimensional images. The latter were generated by taking a z-series of 1-μm-thick optical sections, usually 20–30, throughout the sample, and then reconstructing and editing the images using the three-dimensional reconstruction program VoxelView (Vital Images, Inc.). Freshly purified brain microvessels were incubated with type I heparinase ( Sigma Chemical Co. ; 50 U/ml in DME/F-12; GIBCO BRL ) for 1 h at 37°C under constant, mild agitation. After this time, the reaction was terminated by dilution with DME/F-12 containing 10% calf serum ( GIBCO BRL ), and samples of the microvessels prepared for combined immunohistochemical detection of heparan sulfate and binding with biotinylated chemokines. To determine the significance of the effect of heparinase I treatment on chemokine binding, a one-way ANOVA was performed, followed by a Bonferroni multiple comparisons test. To visualize chemokine binding sites, isolated brain microvessels were reacted first with biotinylated MCP-1 or biotinylated MIP-1α, and subsequently with fluorescein-avidin . The pattern of MCP-1 binding appears as a near continuous sheath encapsulating the length of the vessel fragment. In comparison, the binding of MIP-1α exhibits a discontinuous or punctate arrangement along the microvascular segment. Control experiments, using concurrent exposure of microvessels to labeled chemokines and antichemokine antibodies, revealed no such staining. PBM, which express receptors for both MCP-1 and MIP-1α ( 2 , 3 , 19 , 35 ), also demonstrated binding of both labeled chemokines. No staining of PBM was obtained in the presence of antichemokine antibodies. To better visualize the unique topologic distributions of MCP-1 and MIP-1α binding sites, confocal images of microvessels were reconstructed to display both endothelial cells and chemokine binding in three-dimensional space . As clearly shown in Fig. 2 , the sphere of MCP-1 binding envelopes the endothelial cells in a relatively smooth and nearly continuous pattern. In striking contrast, binding of MIP-1α appears clustered in discrete patches, periodically decorating the abluminal endothelial surface in a punctate fashion. As the binding patterns of biotinylated MCP-1 and biotinylated MIP-1α were clearly distinguishable, experiments were performed to confirm the presence of separate and specific binding sites for these chemokines along the microvascular surface. Specifically, studies were conducted to determine whether binding of these labeled chemokines could be antagonized by increasing concentrations of unlabeled ligand. Fig. 3 unequivocally indicates that such competition can be achieved for both MCP-1 and MIP-1α. Greater than 90% of the binding of both labeled chemokines could be inhibited by competition with 100-fold excess of the respective unlabeled chemokines. Moreover, there was no cross-inhibition between these two chemokines, i.e., unlabeled MCP-1 did not inhibit the binding of labeled MIP-1α, or vice versa. Results from these competition studies further underscore the premise of separate and specific binding sites for MCP-1 and MIP-1α along the abluminal surface of brain microvessels. In addition to exhibiting homologous, but not heterologous, competition, the binding of both labeled chemokines to perivascular domains was shown to be saturable , with K d values of ∼2 nM for MCP-1 and ∼0.5 nM for MIP-1α. Corresponding Hill coefficients were n = 6 and n = 1.1 for MCP-1 and MIP-1α binding, respectively. Taken together with the qualitative depictions, these quantitative data are consistent with the presence of a limited number of spatially and biochemically distinct, high-affinity binding sites for MCP-1 and MIP-1α along the parenchymal face of microvessels from brain. To exclude the possibility that monocyte-derived perivascular macrophages were the source of chemokine binding sites, double-label fluorescence microscopy was performed to resolve macrophage and MCP-1/MIP-1α distributions. It can be clearly seen in Fig. 5 that the binding patterns of both these chemokines and perivascular macrophages are completely distinguishable. Assessment of colocalization of chemokine binding with other perivascular cell types, such as pericytes, mast cells, plasma cells, and smooth muscle cells, which only showed very limited distribution in these microvessels preparations, additionally revealed drastically dissimilar patterns (data not shown). Lastly, as several chemokines have demonstrated affinity for proteoglycans and other extracellular matrix components ( 11 , 45 , 47 , 49 , 50 ), we examined whether MCP-1 and/or MIP-1α binding coincided with three major constituents of the subendothelial matrix: heparan sulfate, collagen type IV, and laminin. Fig. 5 indicates that binding sites for MCP-1 and MIP-1α lie predominantly internal to both heparan sulfate and collagen type IV domains, but appear intermixed with that of laminin. Thus, it seems unlikely that binding to brain microvessels occurs principally via attachment to either collagen type IV or heparan sulfate. However, laminin may be associated in limited context with the binding of both these chemokines. This interpretation is consistent with a recent report describing MCP-1 and MIP-1α induction of mast cell migration across microporous filters coated with laminin, but not with collagen type IV ( 46 ). To further assure that chemokine binding to brain microvessels did not merely reflect attachment to heparan sulfate, a glycosaminoglycan (GAG) to which many chemokines, including MCP-1 and MIP-1α, have been reported to adhere ( 17 , 25 ), binding experiments were also performed on microvessels stripped of heparan sulfate by treatment with heparinase I. As shown in Fig. 6 , such enzymatic treatment removed nearly all perivascular heparan sulfate, as judged by immunofluorescence. Despite this, neither MCP-1 nor MIP-1α binding to microvessels was diminished. On the contrary, slightly heightened binding of both chemokines was observed . It was further observed that microvessel binding of neither chemokine could be blocked by coincubation with 1,000-fold excess of either heparin or heparan sulfate (data not shown). Collectively, these data strongly argue against heparan sulfate, alone, being responsible for MCP-1 and MIP-1α binding to the abluminal surface of brain microvessels. Similar experiments to gauge the effects of enzymatic removal of laminin and collagen (using cathepsins B and D, and collagenase type IV, respectively) also indicated no significant diminution in the binding of either chemokine (data not shown). Insofar as both kinetic and cytological data indicated that both MCP-1 and MIP-1α were binding to high-affinity sites that were closely associated with the endothelium, we further assessed whether brain microvessels express receptors for these chemokines. These receptors, designated by the abbreviation CCR, have been well documented in leukocytes, and their sequences cloned ( 35 ). CCR2 is considered the lone, specific receptor for MCP-1, while both CCR1 and CCR5 are the recognized receptors for MIP-1α. As indicated in Fig. 8 , brain microvessels display immunoreactivity with specific antibodies to each of these receptors. All three antibodies were similarly reactive with PBM (data not shown), consistent with these cells' ability to bind both chemokines (as visualized above). However, it is important to note that microvascular staining with the antireceptor antibodies was not similar to that obtained with anti-CD68 staining of perivascular macrophages , the latter clearly exhibiting immunoreactivity in discrete cellular domains that lie outside the immediate vascular wall. In a contrary manner, antireceptor antibodies decorate the contour of microvessels in a more continuous manner, and apparently bind to sites in close apposition to the abluminal endothelial surface. Thus, antichemokine receptor staining of microvessels may include, but is not restricted to, perivascular macrophages. Specific binding of MCP-1 and MIP-1α to separate domains along the microvessel outer surface was determined according to the following criteria. First, the distribution patterns of MCP-1 and MIP-1α binding were remarkably different from each other. Second, binding of labeled MCP-1 and MIP-1α could each be inhibited nearly 95% by 100-fold excess of their unlabeled homologues, but not by unlabeled derivatives of the other chemokine. Third, binding of both chemokines was saturable. Finally, neither MCP-1 nor MIP-1α binding was observed to colocalize with three major components of the basement membrane or to be quantitatively dependent upon the presence of these constituents, implying a lack of any significant chemokine trapping within the subendothelial matrix. It is important to reemphasize that the chemokine binding sites visualized and characterized here reside strictly on the abluminal surface of the microvessels. These results are thus to be distinguished from descriptions of radiolabeled chemokine binding to the apical surface of cultured endothelial cells, which represents the luminal microvascular surface in situ. In the latter case, chemokine binding has been demonstrated to be largely dependent upon the presence of GAGs ( 17 , 40 , 41 ). Attachment of chemokines to such GAGs is thought to serve the passive role of sequestering chemokines in the luminal space, raising their effective concentration and, thus, their probability of encountering a chemokine receptor on a loosely tethered leukocyte ( 17 ). In contrast to that observed in these previous reports, MCP-1 and MIP-1α binding to isolated brain microvessels was not diminished as a result of enzymatic removal of perivascular heparan sulfate, a prominent chemokine-binding GAG. As similar heparinase treatment to that performed here has been shown to completely antagonize the binding of numerous chemokines to both cultured cells and isolated subendothelial matrix ( 11 , 25 ), our findings infer that, at the very least, attachment to heparan sulfate is not the sole mechanism underlying MCP-1 and MIP-1α binding to human brain microvessels. Of course, these chemokines might interact with GAGs other than heparan sulfate present along the abluminal surface of brain microvessels, and do so with varying affinities ( 49 ). However, the K d values obtained here (≤2.0 nM) are significantly lower than those generally reported for chemokine attachment to various GAGs, e.g., mid-nanomolar to low millimolar range ( 27 ). That enzymatic removal of both laminin and collagen was additionally unable to lessen the degree of MCP-1 and MIP-1α binding sustains the concept that these perivascular chemokine binding domains are more closely associated with integral elements of the brain microvasculature than with components of the subendothelial matrix. The additional lack of coincidence between chemokine binding and the presence of perivascular cells also eliminates these cells as being the major target of chemokine binding. What then might be the nature of these abluminal chemokine binding sites? MCP-1 and MIP-1α binding may reflect attachment to specific chemokine receptors on the surface of endothelial cells. In this regard, the fluorescence competition assays described here mirror those reported for chemokine binding to cloned CCR1 and CCR2 receptors ( 31 , 32 ), in that 100-fold excess of unlabeled ligand abrogated nearly 95% of labeled chemokine binding. That we were further able to detect immunostaining with antibodies to CCR1, CCR2, and CCR5 is yet additional support. Lastly, the estimated K d values for both MCP-1 and MIP-1α binding to brain microvessels are within the low nanomolar range generally reported for these chemokines' binding to their respective receptors (reviewed in 27 , 43 ). The relatively high degree of apparent cooperativity in MCP-1 binding, as indicated by a Hill coefficient of n = 6, may reflect cooperative interactions between high-affinity chemokine receptors and low-affinity extracellular matrix component(s) ( 17 ). Thus, our observations could be the first evidence of expression of receptors for MCP-1 and MIP-1α on, or around, the abluminal surface of microvascular endothelium. In agreement with this contention, recent studies have indicated both expression and activity of chemokine receptors on cultured endothelial cells ( 7 , 8 , 15 ), although no cytological distributions of these receptors were described. Aside from binding to specific receptors on brain microvessels, MCP-1 and MIP-1α may also be engaging the Duffy antigen receptor for chemokines (DARC), a promiscuous receptor that binds several chemokines ( 24 ) and is expressed in endothelial cells ( 33 ). Consistent with this possibility, Horuk et al. ( 18 ) demonstrated immunocytochemical staining of capillaries and postcapillary venules of human brain sections with an antibody to DARC. However, considering the different binding patterns for MCP-1 and MIP-1α reported here, and the noted lack of affinity of MIP-1α for DARC ( 24 ), binding to DARC cannot be the exclusive means by which both these chemokines associate with brain microvessels. What could be the functional significance of these perivascular chemokine binding domains? Middleton et al. ( 29 ) presented evidence that another chemokine, interleukin 8, is transcytosed from the abluminal to the luminal microvascular surface in skin, where it appears to reside on the tips of endothelial microvilli. Such luminal “presentation” of bound chemokines is thought to provide the proper context for efficient activation of leukocyte β-integrins ( 2 , 6 , 44 , 45 ). The binding sites described here might represent domains to which the MCP-1 and MIP-1α initially dock, before transcytosis across the microvascular endothelium of brain. MCP-1 and MIP-1α binding to brain microvessels may additionally signal vascular permeability changes through cytoskeletal reorganization, as has been suggested most recently for interleukin 8 ( 9 ). Furthermore, binding to these abluminal sites may function to prevent these chemokines from being diluted within the perivascular space and, thus, augment the intensity, prolong the duration, and/or influence the site of inflammatory reactions ( 11 ). While all these possibilities remain a priori, the pleiotropic nature of chemokines ( 30 , 43 ) is consistent with these binding sites subserving several functions. Are these perivascular chemokine binding sites restricted to, or enriched in, microvessels from brain? While this remains to be explored, the stringent requirements for maintaining an effective blood-brain barrier dictates that specialized mechanisms, possibly receptors, exist within brain microvessels for the purpose of efficiently communicating chemokine signals. In contrast, less restrictive vascular beds may simply allow tissue-derived chemokines to percolate through leaky endothelial junctions into the vascular lumen. That Randolph and Furie were not able to demonstrate significant binding of MCP-1, when this chemokine was applied to the basolateral (abluminal) surface of cultured monolayers of human umbilical vein endothelial cells ( 36 ), may, in part, reflect this phenotypic diversity. It may also be that specific endothelial chemokine binding sites are rapidly lost, or their membrane polarity altered, upon adaptation to culture conditions ( 39 ). As the myriad of chemokine functions begins to resolve, so will their pathogenic role(s) in disease become more appreciated. In turn, this will lead to greater efforts to specifically antagonize chemokine action at sites of inflammation and infection. Heightened understanding of the interactions of chemokines with their binding sites on the vascular surface will be a significant step in this direction. | Study | biomedical | en | 0.999998 |
10209034 | BSA, formyl-methionylleucylphenylalanine (FMLP), and dextran were purchased from Sigma Chemical Co. Recombinant human tumor necrosis factor (TNF)-α, recombinant human interferon (IFN)-γ (specific activity of 10 7 U/mg), and recombinant interleukin 8 (IL-8) were obtained from R&D Systems, Inc. Fluorescent reagent, 2′,7′-bis-(carboxyethyl)-5,6-carboxy-fluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes, Inc. A recombinant form of the third fibronectin type III repeat of chicken tenascin-C containing alanine substitution mutations within the RGD site (TNfn3RAA), was obtained from Anita Prieto and Kathryn Crossin (Scripps Research Institute, La Jolla, CA) and prepared in Escherichia coli . A recombinant VCAM-1/IgG chimera was produced in baculovirus as previously described. Recombinant intercellular adhesion molecule-1 (ICAM-1)-C κ fusion protein was a gift from B. Imhof (Centre Medicale Universitaire, Geneva, Switzerland) to D. Erle (University of California, San Francisco, CA). Ficoll-hypaque plus for isolation of neutrophils from venous blood was purchased from Pharmacia Biotech, Inc. and used according to the manufacturer's specifications. Mouse mAbs, Y9A2 against human α9β1 and AN100226M against α4 , were prepared as previously described. Mouse mAbs, W6/32 against human MHC and IB4 against the integrin β2 subunit, were prepared from hybridomas obtained from American Type Tissue Collection. Mouse monoclonal antihuman VCAM-1 (CD106) was purchased from R&D Systems. FITC-labeled mouse monoclonal anti-CD16 antibody was purchased from Caltag. Human umbilical vein endothelial (HUVE) cells were purchased from Clonetics and grown in endothelial cell growth media (EGM) containing 2% FBS, 10 ng/ml human recombinant EGF, 50 μg/ml gentamycin, 50 ng/ ml amphotericin B, 12 μg/ml bovine brain extract, and 1 μg/ml hydrocortisone and were used between passage 3 and 10. α9- and mock-transfected SW480 and CHO cells were generated by transfection with the previously described full-length α9 expression plasmid pcDNAIneoα9 or the empty vector pcDNAIneo (Invitrogen Corp.) by calcium phosphate precipitation. Transfected cells were maintained in Dulbecco's minimal essential medium(DMEM) supplemented with 10% FCS and the neomycin analogue G-418 (1 mg/ml; Life Technologies, Inc.). Both cell lines continuously expressed high surface levels of α9β1 as determined by flow cytometry with Y9A2 . Cultured cells were harvested by trypsinization and rinsed with PBS. Nonspecific binding was blocked with normal goat serum at 4°C for 10 min. Cells were then incubated with primary antibodies (unconjugated or conjugated with FITC) for 20 min at 4°C, followed by secondary antibodies conjugated with phycoerythrin (Chemicon International, Inc.). Between incubations, cells were washed twice with PBS. The stained cells were resuspended in 100 μl of PBS and fluorescence was quantified on 5,000 cells with a FACScan ® ( Becton Dickinson and Co.). Cells were lysed in immunoprecipitation buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl 2 , 1% Triton X-100, 0.1% SDS, and 0.1% NP-40) supplemented with 10 μg/ml pepstatin ( Sigma Chemical Co. ), 10 μg/ml leupeptin, 5 μg/ml aprotinin ( Calbiochem-Novabiochem Corp. ), and 1 mM phenylmethylsulfonyl fluoride ( Sigma Chemical Co. ). Human neutrophils (10 7 ) were incubated with 1 mM diisopropyl flurophosphate ( Sigma Chemical Co. ) for 15 min before cell lysis. After preclearing with protein G–Sepharose, the supernatant was incubated with primary antibody for 2 h at 4°C and immune complexes were captured by protein G–Sepharose for 45 min at 4°C. The beads were washed five times, and boiled in 2.5× nonreducing Laemmli sample buffer, and samples were separated by SDS-PAGE on 7.5% gels under reducing conditions and transferred to Immobilon membranes. Membranes were blocked with 4% casein, incubated with affinity-purified anti-α9 cytoplasmic domain antiserum 1057 , and developed with luminol. Wells of nontissue culture treated polystyrene 96-well flat bottomed microtiter plates (Nunc Inc.) were coated by incubation with 100 μl VCAM-1/Ig or TNfn3RAA for 1 h at 37°C. After incubation, wells were washed with PBS, then blocked with 1% BSA in DMEM at 37°C for 30 min. Control wells were filled with 1% BSA in DMEM. SW480 or CHO cells were detached using trypsin/EDTA and resuspended in serum-free DMEM. For blocking experiments, cells were incubated with 10 μg/ml Y9A2 and/ or 100226, for 15 min at 4°C before plating. The plates were centrifuged (top side up) at 10 g for 5 min before incubation for 1 h at 37°C in humidified 5% CO 2 . Nonadherent cells were removed by centrifugation (top side down) at 48 g for 5 min. Attached cells were fixed with 1% formaldehyde and stained with 0.5% crystal violet, and the wells were washed with PBS. The relative number of cells in each well was evaluated after solubilization in 40 μl of 2% Triton X-100 by measuring the absorbance at 595 nm in a microplate reader (Bio-Rad Laboratories). All determinations were carried out in triplicate. For adhesion assays on HUVE cells, confluent monolayers of HUVE cells were prepared in 96-well plates in 250 μl of EGM with 2% FBS. Plates were washed twice with serum-free DMEM, then stimulated for 24 h at 37°C with TNF-α (3 ng/ml) or IFN-γ (3 ng/ml) in serum-free DMEM. SW480 cells were detached using trypsin/EDTA and labeled with 2 μM BCECF-AM at room temperature for 30 min. Then cells were washed three times with serum-free DMEM and incubated with blocking antibody, Y9A2 (10 μg/ml), 100226 (10 μg/ml), or combinations of these antibodies for 15 min on ice. In some experiments, HUVE cells were incubated with CD106 (5 μg/ml) for 15 min at 37°C. 50,000 cells in 200 μl of serum-free DMEM were added to each well, and plates were centrifuged at 20 g for 5 min, and covered with aluminum foil to prevent photobleaching. Plates were then incubated for 60 min at 37°C in 5% CO 2 . After incubation, nonadherent cells were removed by washing twice with serum-free DMEM. Finally, 200 μl of the same medium was added to each well, and fluorescence was quantified with a fluorometer (Fluoroskan II; Labsystems) at excitation wavelength 485 nm and emission wavelength 538 nm. The adherent ratio (%) was calculated as follows: ( fluorescence from experimental sample − fluorescence from negative control sample ) ÷ total fluorescence added to chamber . All determinations were carried out in triplicate. Neutrophils were purified from human peripheral venous blood containing 20 U/ml of heparin. Neutrophils were isolated by ficoll-hypaque density gradient centrifugation, followed by 3% dextran sedimentation . Erythrocytes were subjected to hypotonic lysis, remaining neutrophils were washed and resuspended in PBS. The isolated neutrophils were >95% pure and >95% viable as assessed by Wright-Giemsa staining and trypan blue exclusion, respectively. Cell migration was analyzed essentially as described by Marks et al. . In brief, glass coverslips were placed in 35-mm culture dishes and incubated with 100 μl serum-free media containing 10 μg/ml VCAM-1/Ig, 10 μg/ml TNfn3RAA, and 5 μg/ml of ICAM-1 or 1% BSA for 60 min at 37°C, washed, and then incubated with 1% BSA for 30 min. Neutrophils were incubated with no antibody, Y9A2 (10 μg/ml), 100226 (10 μg/ml), IB4 (20 μg/ml), or combinations of antibodies for 15 min at 4°C, and were then incubated for 10 min at 37°C with or without 10 nM FMLP. 10 4 cells were plated onto the coverslip area of each well and allowed to attach at 37°C for 5 min. Dishes were then placed on a videomicroscope stage and individual fields (200×) were recorded for 3 min. Three different fields were examined in each chamber. To count the number of migrating cells in a given field, outlines were made of each cell. Cells were considered to have migrated when both the leading edge and tail of the cell moved ≥7 μm from their initial position. At least 40 neutrophils were analyzed per field and the ratio of migrating to total cells was calculated. Transendothelial neutrophil migration was assessed as described by Cooper et al. . HUVE cells were plated onto polycarbonate inserts (Transwell, 6.5-mm diameter, 8-μm pore for 24-well plate; Costar Corp.) in 200 μl of serum-containing EGM, and allowed to grow to confluence over 72 h. 500 μl serum-free DMEM was added to the lower chamber of each well. 24 h before addition of neutrophils, upper chambers were washed twice with serum-free media and new medium with or without 3 ng/ml of TNF-α. Immediately before the addition of neutrophils, the upper chambers were washed twice with serum-free DMEM and medium in the lower chamber was replaced with 500 μl serum-free DMEM or serum-free DMEM with 10 nM FMLP or 50 ng/ml IL-8. In some experiments HUVE cells were incubated with CD106 (5 μg/ml) at 37°C for 15 min. Purified neutrophils were incubated with no antibody, Y9A2 (10 μg/ml), 100226 (10 μg/ml), IB4 (20 μg/ml), W6/32 (10 μg/ml), or combinations of antibodies for 15 min at 4°C, and 2 × 10 5 cells in 200 μl of media were added to each upper chamber. After 3 h at 37°C in 5% CO 2 , nonadherent cells in the upper chamber were removed. Medium, including migrated neutrophils from the lower chamber, was collected, the lower chamber was rinsed several times to collect all the neutrophils that had transmigrated, and the absence of additional adherent neutrophils was confirmed microscopically. The medium and all washes were pooled and resuspended, and cells were counted with a hemocytometer. All determinations were carried out in duplicate and repeated at least twice. To determine whether VCAM-1 could function as a ligand for α9β1, we performed cell adhesion assays with two different cell lines, SW480 and CHO, that had been stably transfected with either an α9-expression plasmid or empty vector. Both cell lines stably expressed α9β1 on the cell surface as demonstrated by flow cytometry with the anti-α9β1 antibody Y9A2 . Adhesion assays were performed on plates coated with either the known α9β1 ligand, recombinant TNfn3RAA , or recombinant VCAM-1/Ig . For both cell lines, α9-transfectants adhered to both TNfn3 and to VCAM-1 in a concentration-dependent manner, whereas mock-transfectants did not adhere to either substrate. Adhesion of each α9-transfected cell line was completely inhibited by the anti-α9β1 antibody, Y9A2, demonstrating that this effect was mediated by α9β1. To determine whether α9β1-mediated adhesion to VCAM-1 was biologically significant, we next examined the role of this integrin in adhesion of cells to resting HUVE cells, and to HUVE cells that had been activated by incubation with TNF-α (3 ng/ml), a well characterized inducer of VCAM-1 expression, or IFN-γ (3 ng/ml), a cytokine that does not induce VCAM-1 expression. The effects of each cytokine on VCAM-1 expression under the conditions used in these experiments were examined by flow cytometry with anti–VCAM-1 antibody CD106 . As expected, resting HUVE cells and HUVE cells stimulated with IFN-γ did not express detectable levels of VCAM-1, but VCAM-1 was dramatically induced by TNF-α . All cell lines examined demonstrated baseline adhesion to resting HUVE cells, and demonstrated a similar level of adhesion to HUVE activated by IFN-γ, and this baseline adhesion was unaffected by anti-α9β1 antibody . However, only α9-transfected cells demonstrated enhanced adhesion to TNF-α–treated HUVE. This enhanced adhesion was returned completely to basal levels by antibody to either α9β1(Y9A2) or to VCAM-1 (CD106), demonstrating that it was due to an interaction between α9β1 and VCAM-1. We have previously demonstrated that α9β1 is widely expressed on epithelial and smooth muscle cells , but expression on leukocytes has not been reported. To determine whether α9β1 is expressed on cells likely to encounter activated endothelial cells, we performed flow cytometry on whole blood leukocytes with the α9β1 antibody Y9A2. We evaluated expression on neutrophils, monocytes, and lymphocytes by gating on each population separately, based on differential light scattering. From a separate atopic donor we evaluated expression on eosinophils, which were separated from other leukocytes based on light scattering and the absence of surface expression of CD16. In parallel, we examined expression of the structurally related integrin subunit, α4. α9β1 was not detected on lymphocytes or eosinophils and was expressed at low levels on monocytes . In contrast, α9β1 was highly and uniformly expressed on human neutrophils. As expected, α4 was highly expressed on lymphocytes, monocytes, and eosinophils, but was also detected on neutrophils, albeit at considerably lower levels. Expression of α9 on neutrophils was further confirmed by immunoprecipitation with Y9A2 followed by Western blotting with an affinity-purified antiserum raised against a unique portion of the α9 cytoplasmic domain. A band of 160 kD (appropriate molecular mass for α9) was detected in lysate of human neutrophils after immunoprecipitation with Y9A2, but not after immunoprecipitation with the control antibody R6G9 . To determine whether α9β1 expression on neutrophils was biologically significant, we initially sought to examine static adhesion of neutrophils to dishes coated with either TNfn3RAA or VCAM-1. However, in the absence of antibodies against β2 integrins, neutrophils avidly adhered to all surfaces examined, and in the presence of β2 integrin blocking antibodies, neutrophils could not be induced to adhere to either VCAM-1 or TNfn3RAA by incubation with MnCl 2 , FMLP, phorbol esters, or the β1 activating antibody TS2/16 (data not shown). Therefore, we examined the possible role of α9β1 in another important neutrophil function, cell migration. Migration was examined by counting the numbers of individual neutrophils that migrated on chambers coated with either TNfn3RAA or VCAM-1 in the presence or absence of the activating agonist FMLP (10 nM). In the absence of FMLP, very few neutrophils migrated on either substrate , and antibodies against α9β1, α4, or β2 integrins had no effect. In the presence of FMLP, neutrophil migration was significantly enhanced on TNfn3RAA, an effect that was abolished by antibody against α9β1. FMLP also enhanced neutrophil migration on VCAM-1, and this effect was partially inhibited by antibodies against α9β1 or α4, and completely inhibited by the combination of both antibodies. These data demonstrate a significant role for α9β1 in mediating neutrophil migration on both substrates. Antibody against β2 integrins had no effect on neutrophil migration on FMLP-induced neutrophil migration on TNfn3RAA or VCAM-1. However, as expected, antibody against β2 inhibited FMLP-induced migration on the β2 integrin ligand ICAM-1, whereas antibodies against α9β1 or α4 had no effect . We next sought to determine whether the effect of α9β1 and α4 integrin(s) described above was relevant to an in vitro model of neutrophil extravasation–migration across endothelial monolayers. HUVE cells were grown to confluence on the top side of permeable filter supports and incubated in the presence or absence of TNF-α (3 ng/ml). Purified neutrophils were added to the apical compartment in the presence or absence of FMLP added to the basal compartment. These studies were performed in the absence of blocking antibodies, or in the presence of antibodies against α9β1, α4, β2, VCAM-1, control antibody against MHC, or combinations of these antibodies. As expected, in the absence of blocking antibodies, FMLP greatly increased neutrophil migration into the bottom compartment, and this effect was augmented by pretreatment of HUVE cells with TNF-α . No antibody affected basal migration across unstimulated HUVE cells or FMLP-induced migration across unstimulated HUVE cells . However, antibody against either α9β1 or α4 inhibited the augmented migration induced by TNF-α. Antibody against VCAM-1 was equally effective in inhibiting migration across TNF-α–treated HUVE cells, suggesting that TNF-α augmented transmigration was mediated by an interaction between α9β1 and α4 integrins and VCAM-1. As previously reported, antibody against β2 integrins also partially inhibited transmigration in response to FMLP, but this effect was surprisingly small. Essentially identical results were obtained when IL-8 was used as a chemoattractant in place of FMLP (data not shown). The results of the current study demonstrate that the inducible endothelial cell immunoglobulin family member, VCAM-1, is an effective ligand for the integrin α9β1. This receptor–ligand interaction is sufficient to support adhesion of α9-transfected cell lines to VCAM-1 and to TNF-α–activated HUVE cells, an effect that is mediated by the binding of α9β1 to VCAM-1. Furthermore, α9β1 is uniformly and specifically expressed on normal resting human neutrophils, and mediates both neutrophil migration on a fragment of tenascin-C or VCAM-1 and transmigration of neutrophils across TNF-α–activated endothelial monolayers. Together, these data suggest a previously unsuspected role for α9β1 and VCAM-1 in extravasation of neutrophils at sites of acute inflammation. In addition to α9β1, we found detectable, albeit low, levels of the structurally related integrin α4 subunit on resting human neutrophils. This finding is consistent with several previous reports of α4 expression on neutrophils from a variety of species . Although the level of expression of α4 we detected on human neutrophils was one to two orders of magnitude lower than expression on eosinophils, monocytes, and lymphocytes, this low level expression appeared to be biologically significant, since antibody against α4 partially inhibited migration of neutrophils on VCAM-1 and migration across TNF-activated endothelial monolayers. Recently, α4β1 has been shown to mediate both neutrophil adhesion to VCAM-1 and neutrophil transmigration across fibroblast monolayers . As expected, α4 integrins did not contribute to migration on TNfn3RAA, since this fragment of tenascin is not a ligand for either α4 integrin. Adhesion of activated neutrophils to endothelial cells at sites of inflammation is well known to require the participation of integrins sharing the β2 subunit which bind to two other members of the immunoglobulin family expressed on endothelial cells, ICAM-1 and ICAM-2 . ICAM-1 is constitutively expressed on many epithelia, but expression is dramatically induced by a variety of inflammatory stimuli, including TNF-α. Our data do not address the role of α9β1 or α4 integrins in stable adhesion of neutrophils, since we were not able to maintain adhesion of these cells to any substrate in the presence of β2 integrin blocking antibodies. This effect could be due to a critical role of these integrins in adhesion or to an inhibitory signaling pathway through which antibody-mediated ligation of β2 integrins inhibits the function of other integrins, such as α9β1. However, the mechanisms underlying the subsequent steps in neutrophil extravasation, including detachment from sites of initial adhesion and subsequent migration across the endothelial cell surface and components of the underlying extracellular matrix, are not as well understood. The data in this manuscript suggest, at least in the model system used, that α9β1 and α4 integrins are likely to play important roles. Both integrins could contribute to migration across VCAM-1 expressing endothelial cells and shared ligands such as osteopontin , and α9β1 could be critical for migration across tenascin-C that is present outside the vasculature at sites of inflammation . A role for β2 integrin–independent processes in neutrophil extravasation in vivo has been suggested by several sets of observations, including studies of neutrophil extravasation into the liver in response to endotoxin and neutrophil migration into the alveolar spaces of the lung in response to intratracheal instillation of live bacteria . Recent studies demonstrating neutrophil extravasation into the lungs and peritoneal cavity in β2 integrin knockout mice also demonstrate the importance of mechanisms independent of β2 integrins . The extent to which these events are mediated by α9β1 and/or α4 integrins needs to be determined from in vivo studies. We have recently succeeded in generating mice expressing a null mutation in the α9 subunit gene, but these mice die within 10 d of birth (unpublished observation). However, the development of bone marrow chimeras from this line should allow us to directly examine these questions. In addition to the expression on neutrophils described in this report, α9β1 is widely expressed on muscle cells, surface epithelial cells, and hepatocytes . It is unclear what role, if any, interactions with VCAM-1 might have at these sites. VCAM-1 has also been reported to be expressed on muscle cells under various conditions , so it is conceivable that α9β1/VCAM-1 interactions may be biologically significant in muscle as well. Such an effect could explain the apparent contradiction between reports, based on antibody inhibition, that α4β1/VCAM-1 binding plays a critical role in myotube formation and the normal muscle development of α4 knockout cells in chimeric mice , if the α4 knockout led to a developmentally regulated increase in α9β1 expression. In summary, we have identified VCAM-1 as a novel and biologically significant ligand for the integrin α9β1, have demonstrated that this integrin is expressed on neutrophils and mediates neutrophil migration on two relevant ligands and neutrophil transmigration across activated endothelial monolayers. These findings support a role for α9β1/ VCAM-1 interactions in extravasation of neutrophils at sites of inflammation. | Study | biomedical | en | 0.999996 |
10209035 | Normal (IFN-γ +/+ ), IFN-γ gene–disrupted (IFN-γ −/− ), and IL-4 gene–disrupted (IL-4 −/− ) mice, all on the BALB/c background, were purchased from The Jackson Laboratory . Mice were kept in microisolator cages in animal facilities at The University of Alabama at Birmingham Immunobiology Vaccine Center. Mice were provided sterile food and water ad libitum and were free of microbial pathogens as determined by antibody screening and routine histologic analysis of organs and tissues. All mice used in this study were between 10 and 16 wk of age. Mice were given a solution of TNBS (Research Organics) dissolved in a mixture of PBS, pH 7.2, and then mixed with an equal volume of ethanol for a final concentration of 2% TNBS in 50% ethanol. Enemas were performed on mice anesthetized with ketamine/xylazine with a glass microsyringe equipped with a gastric intubation needle. One series of experiments was performed to compare the sensitivity of IFN-γ +/+ or IFN-γ −/− mice to TNBS colitis. In brief, groups of mice were given TNBS at a dose of 25, 36, or 50 μg/g of body weight on days 0 and 7. The severity of disease was assessed by weight loss, fur ruffling, rectal prolapse, and death. A dose of 36 μg TNBS/g of body weight was chosen and given on days 0 and 7, and tissues and cells were assessed on day 10. One set of experiments was performed with mAbs to IFN-γ or IL-4 to determine the roles of these cytokines in the development of TNBS colitis. In brief, mice were given 1 mg of either rat anti–IFN-γ (XMG 1.2) or anti–IL-4 (11B11) mAbs by the intraperitoneal route on the same days as the TNBS enema (days 0 and 7). Control groups received an intraperitoneal dose of normal rat IgG (Jackson ImmunoResearch Labs, Inc.) along with a TNBS enema. The colon was removed from its mesentery to the pelvic brim by blunt dissection. The pelvis was severed, and the rectum was carefully removed from the sacral lymph nodes (SLN) and adjacent tissue. The distal half of the colon was opened longitudinally and fixed in 5% glacial acetic acid in ethanol (vol/vol). After embedding in paraffin, 4-μm-thick serial sections were prepared and stained with hematoxylin and eosin for histologic grading. Thickness of lymphoid follicles was determined on sections that contained follicles extending from the mucosal layer to the serosa using a micrometer. Histologic grading was done blindly according to the criteria listed in Table I . A maximum score of 8 indicated severe colitis with acute ulcers and an overall diffuse pattern of chronic changes. Tissues were freshly frozen in Tissue-Tec OCT compound (Miles-Yeda, Inc.). 7-μm cryostat sections were fixed in ice-cold acetone for 10 min, dried, and rehydrated in Tris-buffered saline. This step was followed by blocking with Tris-buffered saline containing a 1:50 dilution of Fc-blocking 2.4G2 mAb ( PharMingen ) and 10% heat-aggregated rabbit serum ( Sigma Chemical Co. ). Sections were stained for 1 h in the same buffer with PE–anti-CD4 mAb (1:50 dilution; PharMingen ) and biotinylated anti-B220 mAb (1:50 dilution; PharMingen ). These sections were washed with buffer and then stained with streptavidin–FITC (Southern Biotechnology Associates) for 30 min. Reactivity with peanut agglutinin (PNA) was demonstrated using a 1:50 dilution of biotinylated PNA (Vector Labs, Inc.) and streptavidin–FITC. The sections were mounted and viewed using 100× optics and a dual red/green filter. Images from each staining were analyzed for red and green fluorescence using identical settings in Photoshop 4.0 (Adobe Systems, Inc.). For transmission electron microscopy, the colon was prefixed with cold 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h and washed extensively with 0.1 M phosphate buffer. The tissues were then post-fixed with osmium tetraoxide, dehydrated, and embedded in Epon. Sections were cut and examined with an electron microscope. Peyer's, cecal, and colonic patches were excised from the intestinal wall. The colonic lymphoid follicles in naive mice were identified with a magnifying lens, and ∼3–5 follicles/colon were seen. Patches and follicles were washed once with RPMI 1640 (Cellgro Mediatech) and dissociated with collagenase (type IV; Sigma Chemical Co. ) at a concentration of 0.5 mg/ml in RPMI 1640 with 100 U/ml penicillin, 100 μg/ml streptomycin, and 40 μg/ml gentamicin for 20 min at 37°C to obtain single-cell preparations ( 23 ). The cell dissociation was repeated two additional times in fresh collagenase solution each time. The single-cell suspensions were then pooled and washed with RPMI 1640 twice more. Mononuclear cells were further purified using a discontinuous Percoll gradient to avoid contamination with epithelial cells. The SLN were teased with forceps, and the resulting cell suspension was washed with RPMI 1640 two additional times. Colonic lamina propria lymphocytes were prepared as previously described with modifications ( 24 ). In brief, after excision of all visible lymphoid follicles, colonic tissue was treated with 1 mM EDTA in PBS for 20 min to remove the epithelium. The tissue was then digested with collagenase (type IV; Sigma Chemical Co. ) for 20 min, and this step was repeated two more times. The isolated cells were further purified and separated from epithelial cells by centrifugation through a Percoll gradient as described elsewhere ( 24 ). The isolated lamina propria lymphocytes were >99% viable. In these studies, isolated lymphoid cells were stained for various membrane receptors with FITC-, PE-, or biotin-labeled mAbs. Lymphoid cells were first preincubated with an Fc-blocking mAb (clone 2.4G2; PharMingen ) at a concentration of 12.5 μg/ml for 5 min on ice. The cells were then incubated with FITC-, PE-, or biotin-labeled antibodies for 30 min in ice. Biotinylated antibodies were detected with PE– or FITC–streptavidin. The following conjugated or unconjugated anti–mouse antibodies were used: anti-CD3 (clone 145-2C11), anti-CD8 (clone 53.6.7), anti-B220 (clone RA3-6B2), anti–TCR-β (clone H57-597), anti–TCR-γ/δ (clone GL3), anti-IgD (11–26c.2a), anti-IgM (clone II/41), anti-IgA (clone R5-140), and anti–I-A d (clone AMS-32.1) ( PharMingen ). Goat anti–IgG (Fab′) fragment was purchased from Southern Biotechnology Associates. Two-color analysis was performed with a FACStar PLUS® ( Becton Dickinson ). In some experiments, cells were stained with FITC-labeled anti-CD4 mAb (clone GK1.5; University of Alabama at Birmingham Core Facility) and subjected to sorting with a FACStar PLUS® to obtain purified CD4 + T cells (>99% CD4 + T cells). For stimulation of antigen-specific T cells, cells were coupled to TNBS. In brief, cells isolated from patches, lymph nodes, or lamina propria were treated with 0.3 mg/ml TNBS in RPMI 1640 for 15 min at room temperature. Cells were then extensively washed and cultured in RPMI 1640 supplemented with 10% FCS, sodium pyruvate, l -glutamine, Hepes, 50 μM 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamicin, and 1 μg/ml amphotericin B (complete medium) at 37°C in an atmosphere of 5% CO 2 in air. For the proliferation assay, 2 × 10 5 cells were placed in each well of 96-well plates, and 0.5 μCi/well of tritiated [ 3 H]thymidine was added 18 h before harvesting. Peyer's, cecal, and colonic patch cells were incubated for 4 d, and SLN cells were incubated for 3 d. The amount of [ 3 H]thymidine incorporation was determined by scintillation counting. Cells cultured without TNBS treatment were used as controls. In some experiments, TNBS-treated cells were cultured in complete medium for 24 h at a concentration of 3 × 10 6 cells/ml. Culture supernatants were subjected to cytokine-specific ELISA. For mRNA analysis, nonadherent cells were harvested after 24 h of incubation, and CD4 + T cells were purified by flow cytometry. Extracted total RNA was subjected to RT-PCR to assess cytokine-specific mRNA. The CD4 + T cells purified from lamina propria lymphocytes isolated from the colon were subjected to RT-PCR. The details of this assay have been described previously ( 24 – 26 ). In brief, Immunoplates (Nunc MaxiSorp; Nunc, Inc.) were coated with antibodies to individual cytokines and incubated overnight at 4°C. After blocking with 3% BSA in PBS at 37°C for 2 h, diluted samples were added to wells and incubated overnight at 4°C. The wells were then washed and incubated with detecting antibodies, and bound antibody was detected by peroxidase-labeled antibiotin mAb (Vector Labs). 3,3′,5,5′-tetramethylbenzidine was used as a substrate for peroxidase. The following anti-cytokine antibodies for coating or biotinylated antibodies for detection, respectively, were used in this ELISA: anti–IFN-γ, R4-6A2 and XMG 1.2 mAbs; anti–IL-2, JES6-1A12 and JES6-5H4 mAbs; anti–IL-4, BVD4-1D11 and BVD6-24G2 mAbs; anti–IL-5, TRFK-5 and TRFK-4 mAbs; anti–IL-6, MP5-20F3 and MP5-32C11 mAbs; and anti–IL-10, JES5-2A5 and JES5-16E3 mAbs. The ELISA assays were capable of detecting 0.02 ng/ml IFN-γ, 0.05 U/ml IL-2, 1.0 pg/ml IL-4, 0.1 U/ml IL-5, 0.1 ng/ml IL-6, and 0.04 ng/ml IL-10. Total RNA fractions were prepared from antigen-stimulated CD4 + T cells by the acid guadinium–thiocyanate, phenol–chloroform extraction method ( 27 ). Cytokine-specific RT-PCR was performed as previously described in detail with minor modifications ( 28 ). In brief, a standard protocol was used for the RT reaction, and the resultant DNA was amplified by repeating PCR for 35 cycles at 95°C for 1 min and at 60°C for 1 min. Statistical significance was determined by the Mann– Whitney U test using the Statview-J 4.11 statistical program (Abacus Concepts, Inc.) for Macintosh computers. To analyze survival distributions, the Mantel–Cox test was performed, and the significance level chosen was P = 0.05. Previous studies have provided compelling evidence that the Th1-type cytokine IFN-γ plays a major role in experimental IBD in mice. To examine the possible significance of Th2-type responses, we first queried if colitis would develop in mice deficient in IFN-γ production. In these studies, we found that weight loss, colitis, and even death were dependent upon the dose of TNBS given, though the differences in colitis-inducing versus lethal doses were small in normal IFN-γ +/+ mice. Interestingly, IFN-γ −/− mice were more resistant to TNBS–ethanol enemas, with an approximate LD 50 dose for IFN-γ +/+ mice only causing the death of 25% of IFN-γ −/− mice . At the intermediate dose of 36 μg TNBS/g weight, >90% of mice in both groups survived. In both groups, autopsy showed large necrotizing ulcer rings and obstructed colons. With the low dose of TNBS (25 μg/g of weight), all mice survived without symptoms of colitis. We used the intermediate dose of TNBS to compare wasting disease in IFN-γ −/− and IFN-γ +/+ mice . Administration of ethanol only had little effect on body weight and produced no symptoms of colitis in mice of either group. On the other hand, the intermediate dose of TNBS (36 μg/g) together with ethanol induced significant weight loss and diarrhea in both mouse groups . Of interest was the finding that treatment of IFN-γ +/+ mice with anti–IFN-γ mAb did not prevent development of wasting disease . These findings show that although IFN-γ −/− mice were more resistant than IFN-γ +/+ mice to TNBS–ethanol enemas, they did develop significant TNBS-induced wasting disease in the absence of IFN-γ. The pathological features of TNBS colitis were essentially the same in IFN-γ −/− and IFN-γ +/+ mice given TNBS enemas of 36 μg/g. The entire colonic wall became thick from edema. The major colitis lesion was observed in the distal half of the colon, and focal ulcers were detected in ∼70% of colonic tissues from either IFN-γ −/− or IFN-γ +/+ mice. The ulcers often penetrated the colon and adhered to adjacent tissues. Distortion of crypts, loss of goblet cells, and infiltration of mononuclear cells were observed in all mice, and most IFN-γ −/− mice showed these changes in a more extended area of the colon than did IFN-γ +/+ mice. Some parts of the mucosal layer lost crypts and were replaced with lymphocytes, macrophages, and fibrotic tissue . Crypt abscesses were less frequent. Another major finding in this model was an enlargement of lymphoid follicles in the colon. The inflamed colon developed white patches that resembled Peyer's patches in the small intestine and protruded from the colonic wall into the mesenteric side. When examined microscopically, these white patches, which were distinct from other parts of the colonic wall, were found to be lymphoid follicles . Before TNBS enema, the colonic lymphoid follicles were difficult to detect macroscopically but were microscopically identified in tissue sections of both IFN-γ −/− and IFN-γ +/+ mice . When TNBS colitis was induced, ∼7–8 white follicles per mouse were visible and thicker than colonic follicles observed in mice before TNBS enema (Table II ). The cell yield from colonic patches was dramatically increased after induction of colitis, and the follicles in IFN-γ −/− mice contained higher numbers of mononuclear cells than did the follicles in IFN-γ +/+ mice (Table II ). This finding indicates that the enlargement of these follicles in the inflamed colon was actually enhanced by the absence of IFN-γ. We next investigated whether the follicles that appeared after TNBS enema were secondary lymphoid aggregates or enlarged GALT, e.g., Peyer's patch–like structures. As IFN-γ −/− mice developed larger follicles than control mice, we considered it possible that there was a difference in lymphocytes between IFN-γ −/− and IFN-γ +/+ mice that could account for the severity of the disease. Immunohistochemical analysis showed that these follicles had distinct T cell areas and B cell zones with germinal centers . Flow cytometry analysis of mononuclear cells from these follicles indicated that T cell subsets were similar to those of Peyer's patches in the small intestine and to cecal patches. In IFN-γ −/− mice, CD4 + T cells were significantly less frequent in Peyer's or cecal patches. Increases in macrophages or other inflammatory cell types were not observed in colonic patches. When compared with naive mice, both IFN-γ +/+ and IFN-γ −/− mice tended to show decreased percentages of CD4 + T cells (Table III ). On the other hand, the percentage of B cell subsets was slightly elevated in both groups of mice after induction of colitis (Table IV ). Colonic follicles in IFN-γ −/− mice exhibited higher numbers of B cells when compared with either cecal or Peyer's patches. IFN-γ −/− mice had more surface IgA–positive B cells than IFN-γ +/+ mice. The higher percentages of B cells noted in IFN-γ −/− when compared with IFN-γ +/+ mice were also observed before induction of colitis. Collectively, the colonic follicles of mice with TNBS colitis showed a dramatic expansion of both T and B cells that was accompanied by a relative increase in all B cell subsets. M cells were identified in the epithelial cells covering the follicles by electron microscopy. M cells exhibited irregular microvilli with attached bacteria as well as interlaced macrophages and lymphocytes that extended into the mitochondria- and vesicle-rich cytoplasm . M cells were found in follicles of both IFN-γ −/− and IFN-γ +/+ mice. From these studies, we conclude that the lymphoid follicles induced by TNBS colitis are colonic patches that retain an overall structure similar to that of Peyer's patches of the small intestine. The number of patches found in mice with colitis was comparable to numbers reported in naive adult BALB/c mice, as determined by an enumeration method consisting of fixation and illumination of the colon ( 29 ), leading us to conclude that these patches were not induced de novo but were instead enlargements of existing patches which became visible after TNBS enema. Antigen (TNBS)-specific responses of colonic patch cells were examined. Mononuclear cells isolated from colonic patches and SLN of mice with TNBS colitis responded to stimulation with TNBS, whereas cells from Peyer's or cecal patches or colonic lamina propria did not undergo TNBS-specific responses . Cells from SLN of IFN-γ −/− mice had lower proliferative responses than did those of IFN-γ +/+ mice, perhaps due to the lack of response of cells normally activated by the presence of IFN-γ. These results show that TNBS-responding cells are localized in the colonic patches of both groups of mice. Cytokine production accompanied by TNBS-stimulated proliferative responses was also measured (Table V ). Cells from colonic patches and SLN of IFN-γ +/+ mice produced both IL-4 and IL-5 in addition to IFN-γ and IL-2, and the former also produced IL-6. Colonic patch and SLN cells from IFN-γ −/− mice produced IL-4, IL-5, and IL-10. Secretion of IL-5 by cells from IFN-γ −/− mice was higher than that by cells from IFN-γ +/+ mice, and this difference reached statistical significance in cells from SLN. Although it has been shown that Th2-type cytokine responses can be downregulated by IFN-γ ( 30 ), as much or more IL-4 was produced in IFN-γ +/+ mice as in IFN-γ −/− mice. Cytokine levels were also assessed in culture supernatants of lamina propria lymphocytes; however, low levels were seen with or without TNBS stimulation. These results indicate that Th2-type cytokines were produced by colonic patch T cells in the inflamed colon and SLN obtained from both IFN-γ +/+ and IFN-γ −/− mice. To confirm this, purified CD4 + T cells were subjected to analysis of cytokine-specific mRNA by RT-PCR . The expression of mRNA from cytokines in TNBS-specific CD4 + T cells was similar to the results obtained for secreted proteins. In addition, mRNA for IL-13, another Th2-type cytokine, was also detected by RT-PCR. In additional studies, we examined the time course of mucosal cytokine responses in IFN-γ +/+ and IFN-γ −/− mice. Lamina propria CD4 + T cells were isolated on days 0, 1, and 10 and subjected to cytokine-specific RT-PCR. On day 1, ulcers were present in the colon; however, the colonic patches were poorly visible, and the size of SLN was much smaller than that observed on day 10. In IFN-γ +/+ mice, lamina propria CD4 + T cells obtained on days 0, 1, or 10 did not express IFN-γ mRNA, although the total lamina propria cell population contained IFN-γ on days 1 and 10 . Furthermore, freshly isolated CD4 + T cells taken from colonic patches or SLN on day 0, 1, or 10 also failed to express mRNA for IFN-γ. mRNA specific for IL-4 was detected in CD4 + T cells from lamina propria on day 10 in both groups of mice . When colonic lamina propria CD4 + T cells isolated from IFN-γ +/+ or IFN-γ −/− mice were stimulated with anti-CD3 mAbs, mRNA for both IL-4 and IL-5 was detected by RT-PCR (data not shown). Thus, it was shown that both IFN-γ +/+ and IFN-γ −/− mice with TNBS colitis exhibit Th2-type cytokine responses in colonic patches, SLN, and lamina propria. Our results to this point have shown that Th2-type, rather than Th1-type cytokines, are important in the lymphoid tissues of the inflamed colonic site. We next studied TNBS colitis in the absence of IL-4. Anti–IL-4 mAb treatment failed to prevent the initial weight loss after TNBS enema in normal IL-4 +/+ mice. IL-4 −/− mice given TNBS also suffered from severe wasting disease and died within 3 d at the same rate (10%) as untreated mice. However, the colitis in anti–IL-4 mAb-treated or in IL-4 −/− surviving mice was different from that in IFN-γ +/+ or IFN-γ −/− mice . IFN-γ −/− mice showed higher histologic scores than control mice. On the other hand, IL-4 −/− mice as well as normal mice treated with anti–IL-4 mAb generally exhibited mild lesions, especially in terms of deformation of crypts and chronic changes that resulted in significantly lower histological scores. Taken together, our present data indicate that Th2 cells and the cytokine IL-4 play important roles in the induction of crypt inflammation and hypertrophy of colonic patches in the chronic phase of TNBS colitis induced in BALB/c mice. Two major new findings emerged from this study. Our studies have provided the first evidence that TNBS colitis develops in the absence of IFN-γ in BALB/c mice. In addition, the TNBS colitis lesion is an enhanced expansion of colonic patches where Th2-type cytokine responses are prominent. The enlargement of colonic patches in TNBS colitis was more evident in Th1-deficient IFN-γ −/− mice than in IFN-γ +/+ mice. These findings indicate that Th2-type responses derived from colonic patches play a significant role in TNBS colitis without a requirement for IFN-γ. Hypertrophy of patches in the inflamed colon appears to be due to localized recurrent antigenic stimulation from luminal TNP-haptenated antigens. Other GALT, e.g., cecal and Peyer's patches, did not respond to TNBS, suggesting that colonic patch cells were responding to locally derived luminal antigens. Although the abundant presence of colonic patches in humans has been well documented ( 31 , 32 ), there are no studies reporting a direct relationship between IBD and colonic lymphoid tissues. However, we would certainly expect that antigen uptake and processing by colonic patches would be important during the process of inflammation. In this regard, it has been reported that the abnormal immune responses to intestinal flora that follow a loss of tolerance may be one of the mechanisms for IBD in humans ( 33 , 34 ) and in mouse models ( 35 , 36 ). Because colonic patches are more exposed to bacterial antigens than are the Peyer's patches of the small intestine, the immunoregulatory functions of colonic patches as mucosal inductive sites are important in the pathogenesis of IBD. Recent clinical studies have shown different cytokine profiles in Crohn's disease and ulcerative colitis. In Crohn's disease, the production of IFN-γ and the number of IFN-γ–producing cells were increased ( 18 – 20 ), whereas in ulcerative colitis, the increase was less pronounced. Lamina propria CD4 + T cells from Crohn's disease patients showed increased production of IFN-γ after stimulation via the CD28/CD2 accessory pathway, whereas cells from ulcerative colitis patients showed enhanced production of IL-5 but not IFN-γ ( 21 ). Furthermore, upregulation of IL-12 was observed with Crohn's disease but not with ulcerative colitis ( 22 ). Thus, it is generally recognized that Th1-type T cells are mainly involved in Crohn's disease, whereas Th2-type responses may have a more significant role in ulcerative colitis. To this end, our findings support the notion that Th2-type cells are involved in the development of an intestinal inflammation characterized by diffuse damage to the mucosal layer that bears resemblance to ulcerative colitis. A high dose of TNBS enema induced deep ulcers in the acute phase of colitis that led to rapid weight loss and early death in IFN-γ +/+ mice. On the other hand, IFN-γ −/− mice were more resistant to this initial lethal phase of the disease. These results suggest that IFN-γ is important in the acute phase of the disease. This notion was supported by the presence of IFN-γ in total lamina propria lymphocytes from the early phase of disease in IFN-γ +/+ mice . However, lack of mRNA for IFN-γ in CD4 + T cells indicates that the production of IFN-γ was likely induced in other cell types that comprise innate immunity, rather than by TNBS-specific Th1-type T cells in this experimental system. Lesions observed in the later phase of colitis induced by the intermediate dose of TNBS included distortion of crypts, loss of goblet cells, and infiltration by mononuclear cells. These lesions were frequently found to be accompanied by a total loss of crypts with fibrosis and the presence of a small number of granulomas. These morphological changes in crypts parallel those in ulcerative colitis. Although both IFN-γ +/+ and IFN-γ −/− mice showed these crypt changes with Th2-type responses, the histological score in IFN-γ −/− mice was somewhat higher than in IFN-γ +/+ mice . Furthermore, IFN-γ −/− mice developed larger colonic patches with higher numbers of B cells than did IFN-γ +/+ mice, which was due in part to increased Th2-type responses. The lesions that resulted in all of these mouse groups included enlargement of colonic patches. These findings indicate that Th2-type responses play an important role in chronic inflammation associated with TNBS colitis. In this regard, we were unable to detect mRNA for IFN-γ in CD4 + T cells isolated from the colonic lamina propria throughout the course of disease; however, we did detect mRNA for IL-4 on day 10. TNBS colitis induced in SJL/J mice ( 14 ) or other models of colitis ( 4 , 10 , 12 ) where Th1-type responses are predominant are characterized by a dense cell infiltration with active destruction of all layers throughout the entire colon. In contrast, the inflammation that was induced in IFN-γ +/+ or IFN-γ −/− BALB/c mice on day 10 was characterized by atrophic changes of the mucosal layer in the distal colon, which occurred in the absence of extensive lymphocyte infiltration or destruction of tissue. Thus, histological features in our model likely explain the lack of antigen-specific proliferative responses and cytokine production by lamina propria lymphocytes. We postulate that this is a feature of Th2-type inflammation in response to antigen that occurs in the inductive site itself, e.g., the colonic patches where constant stimulation of Th2-type cells disturbs the normal tissue repair processes in lamina propria and regeneration of epithelial cells, leading to fibrosis and the deformity seen in crypts. Further support for this concept was offered by our finding that IL-4 −/− and normal BALB/c mice treated with anti–IL-4 mAb developed an initial wasting disease and severe ulcers but exhibited a milder lesion than did control mice in the chronic phase of the disease. Our study demonstrated that TNBS colitis in IFN-γ +/+ mice was similar to Th2 cytokine-mediated colitis observed in IFN-γ −/− mice. This is not surprising, as BALB/c mice are known to favor Th2-type development of T cells, whereas other mouse strains, such as B10.D2, favor Th1-type development ( 37 , 38 ). In this regard, the colonic patch cells from IFN-γ +/+ mice tended to produce larger amounts of IL-4 and IL-6 when compared with identical cell fractions taken from IFN-γ −/− mice. The low production of IL-6 in cell cultures from IFN-γ −/− mice is most likely due to insufficient activation of macrophages in cultures lacking IFN-γ. We also consider the decreased IL-4 responses in IFN-γ −/− mice to be due to insufficient cell activation, as determined by proliferation assays. These findings suggest that upregulation of IL-4 production associated with TNBS colitis in BALB/c mice may be independent of a cytokine regulatory network involving IFN-γ. Evidence for a Th2-type response with colitis has been reported in TCR-α–deficient mice, which develop spontaneous colitis. In these mice, cytokine production by mesenteric lymph node cells showed a marked increase in IL-4 synthesis ( 5 ). Furthermore, studies have also shown that local CD4 + TCR-α − /β dim+ T cells were responsible for production of IL-4 ( 8 ). Recent studies of hapten-induced colitis using oxazolone have also shown that Th2-type cells can mediate intestinal inflammation ( 39 ). Thus, it is possible that Th2-type responses are also involved in other mouse models of IBD. Previous studies by others have emphasized the major importance of IFN-γ production by Th1-type T cells in TNBS colitis in a different mouse strain (SJL/J; 14). In contrast, our study has now shown that TNBS colitis is associated with Th2-type responses and with a pattern of inflammation that resembles ulcerative colitis. Thus, the immune responses to intracolonic TNBS can induce either Th1- or Th2-type responses, which are associated with distinct types of colitis. The apparent differences in experimental conditions between our studies and those of others are in the mouse strain and the dose of TNBS ( 14 ). However, luminal components such as food antigens or intestinal microflora may also be involved. These congenital and acquired factors could also contribute to the differentiation of phenotypes of T cell responses and colonic inflammation. It is also clear that there are clinical cases of IBD that are difficult to diagnose as either Crohn's disease or ulcerative colitis. Furthermore, the phenotype of disease can shift from one to the other during disease progression. It is possible that environmental factors influence induction of Th1- or Th2-prone responses that reflect distinct pathological features. Further investigation of the phenotypic shift in TNBS colitis using normal and cytokine-deficient mice will contribute to a better understanding of the complex pathogenesis of IBD and may reveal the underlying immunologic differences that give rise to Crohn's disease and ulcerative colitis. | Study | biomedical | en | 0.999996 |
10209036 | Purification of gp30/40, in-gel digestion, nanoelectrospray tandem mass spectrometric sequencing, and assembly of peptide sequence tags for database searching have been described previously ( 20 , 23 – 26 ). Peptide esterification was performed on a part of the unseparated digest by treatment with 2 M HCl in aqueous-free methanol for 45 min at room temperature. After derivatization, the reaction mixture was dried in a vacuum centrifuge. Based on an expressed sequence tag (EST) cDNA sequence corresponding to the (YSEVV(L/I)DSEPK) peptide obtained by nanoelectrospray sequencing of purified pp29/30, forward and nested forward oligonucleotides (CCTGCCAGGGCTGCAGAGGAGGTG and CCCCGAGCCGGAGCTCTATGCCTC) and reverse and nested reverse oligonucleotides (TGCGGGTCTGGGCACATACTGAGGC and CACCTCCTCTGCAGCCCTGGCAGG) were used for 3′- and 5′-rapid amplification of cDNA end (RACE) reactions as previously described ( 20 ). The resulting products were purified from agarose gels and sequenced directly. 5′- and 3′-specific primers corresponding to the deduced untranslated regions were used to amplify the complete cDNA, which was double-strand sequenced. For cloning of murine gp30/40, a partial cDNA was generated using 5′ and 3′ primers (CAGCAACTTTGACACTGTCAGTG and ATCCGACCTTGAGCTGGTCGC, respectively) deduced from a mouse gp30/40 EST clone. The fragment was then used for screening a mouse genomic library (provided by Dr. K. Pfeffer, Technical University, Munich, Germany). Resulting clones were sequenced and primers specific for the putative 5′ and 3′ untranslated regions were used to amplify murine gp30/40 from mouse thymocyte cDNA. The resulting fragment was gel-purified and double-strand sequenced. All sequencing reactions and computer-assisted sequence analysis were performed as previously described ( 20 ). The coding region of human wild-type gp30/40 was amplified from HPB-ALL cells cDNA using 5′ and 3′ specific forward and reverse primers (CCCTCGAGCTATGAACCAGGC TGACCCTCGGC and CCTCGAGCTCTACGGGGGGCTGGGGCAGTG, respectively). The purified fragment was cloned into the expression vector pEF-BOS ( 27 ). The chimeric CD8–gp30/40 construct (CCG chimera) consists of the extracellular and transmembrane regions (aa 1–145) of CD8α fused to residues 65–196 of human gp30/40 . It was generated by separately amplifying the cDNA encoding the extracellular and transmembrane domains of CD8α using a CD8α-specific 5′ primer (ATGGCCTTACCAGTGACCGCCTTG), a chimeric 3′ primer (GCTCCGGCCCCTGGTCCACTGGGACAAGTGGTTGCA-GTAAAGGGTGATA ACCAG) and the gp30/40 fragment with an overlapping 5′ primer (GCTCCGGCCCCTGGTCCACTGGGACAAGTGGTTGCAGTAAAGGGTGATAACCAG) and a gp30/40-specific 3′ primer (see above). The two overlapping fragments were used as templates for a chimeric PCR with CD8α-5′ and gp30/40-3′ primers. Mutants of gp30/40 and the CCG chimera were produced by site-directed mutagenesis using the Quick Change site-directed mutagenesis kit (Stratagene) according to the manufacturer's procedure. The following mutants were used in this study: gp30/40-N→ Q (N 26 to Q), gp30/40-ITIM (Y 148 to F), gp30/40-STOP (where a FLAG-tag coding sequence followed by a stop codon was inserted after H 62 ), CC-ITIM (Y 148 to F), and CC-STOP (S 71 to stop codon). The sequence of all constructs was confirmed by double-strand sequencing. Human multiple-tissue Northern blots ( Clontech ) were probed with full-length radiolabeled cDNA of gp30/40 under stringent conditions according to the manufacturer's instructions. Cells were cultured in RPMI 1640 supplemented with 10% FCS, 1% penicillin-streptomycin, and 2% glutamine ( GIBCO BRL , Germany). The Jurkat variant CCG (stably expressing the CCG chimera) was established as described elsewhere ( 20 ). The Jurkat variant J.HM1.2.2 ( 27 – 30 ), which is stably transfected with the human muscarinic receptor type 1, was provided by Dr. Arthur Weiss, University of California at San Francisco, San Francisco, CA, with the authorization of Genentech Inc. , and was cultured under standard conditions. The polyclonal antiserum directed at gp30/40 was generated by immunizing rabbits with a KLH-coupled synthetic peptide corresponding to aa 96–114 of gp30/40. Affinity purification of the antiserum was performed as previously described ( 31 ). SHP2, MAP kinase, PTYR (4G10) Abs (Upstate Biotechnology Inc.), myc (9E10, a gift from Dr. D. Cantrell, Imperial Cancer Research Fund, London, UK), and FLAG (M2, Kodak ) Abs were used at 1 μg/ml for Western blotting, and rabbit p59 fyn and p56 lck antisera (provided by Dr. A. Veillette, McGill Cancer Center, McGill University, Montreal, Canada) were diluted 1:2,000 (vol/vol). CD3-ε (OKT3, IgG2a) or TCR (C305, IgM, provided by Dr. A. Weiss) mAbs were used at 5 μg/ml or as hybridoma supernatant, respectively. The polyclonal antiserum directed at lymphocyte phosphatase–associated phosphoprotein has been described previously ( 31 ) and was used at a 1:5,000 dilution for Western blot analysis. For immunoprecipitation experiments, protein A–Sepharose-purified PTYR (PY72, provided by Dr. B. Sefton, Salk Institute, San Diego, CA), CD3-ε, or CD8 (AICD8.1, IgG1) mAbs were covalently coupled to cyanogen bromide–activated Sepharose beads (6 mg/ml packed beads). Typically, 15 μl of packed coupled beads were used for immunoprecipitation. Cells were activated with culture supernatant of C305 mAb or a combination of biotinylated CD3-ε plus CD4 Abs (10 μg/ml each) followed by cross-linking with avidin (80 μg/ml), PHA (1 μg/ml), 0.1 mM Na Vanadate plus 1 mM H 2 O 2 at 37°C. Immunoprecipitations were performed using gp30/40 antiserum (1:100 vol/vol dilution) followed by protein A–Sepharose, or using CD8, CD3, or anti-PTYR mAb-coupled beads, as described elsewhere ( 20 ). Alternatively, the isolated SH2 domain of SHP2 (provided by Dr. B. Neel, Beth Israel Deaconess Medical Center, Boston, MA), was expressed as glutathione- S -transferase (GST) fusion protein and used for precipitation experiments as previously reported ( 17 , 32 ). After washes, the individual precipitates were subjected to SDS-PAGE and further processed by Western blot analysis. In vitro kinase assay, reprecipitation of in vitro phosphorylated proteins, two-dimensional gel electrophoresis, and Western blot analysis were performed as previously described ( 20 , 33 ). In vitro–labeled gp30/40 was obtained from a primary CD3 immunoprecipitate by reprecipitation using gp30/40 antiserum as reported elsewhere ( 31 ). The reprecipitated material was subjected to SDS-PAGE and the position of SIT was determined by autoradiography. The corresponding band was excised from the dried gel, electroeluted, precipitated with acetone, and subjected to deglycosylation using endoglycosidase F ( Boehringer Mannheim ) as previously described ( 34 ). Jurkat cells (7.5 × 10 7 ) were stimulated with 0.1 mM pervanadate plus 1 mM H 2 O 2 for 2 min at room temperature. After one wash in cold TBS, cells were resuspended in 1 ml of hypotonic buffer (50 mM Tris-HCl, pH 7.5, 10 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM vanadate, 1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) and left on ice for 45 min. After sonication, cells were spun down for 15 min at 15,000 rpm and 4°C. Supernatant (= total lysate) was adjusted to 5 ml with hypotonic buffer and centrifuged for 40 min at 33,000 rpm and 4°C using a SW55Ti rotor. The supernatant (= cytosol) was collected, supplemented with detergent and NaCl to reach final concentrations of 1% NP-40 and 150 mM NaCl, respectively, and further processed for anti-gp30/40 immunoprecipitation. The pellet (= membrane fraction) was washed in 5 ml of high salt buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM vanadate, 1 mM PMSF, 1 μg/ml aprotinin, and leupeptin), solubilized in lysis buffer containing 1% NP-40, and subjected to anti-gp30/40 immunoprecipitation as previously described. The confocal laserscan microscopy of HPB-ALL cells and Jurkat cells was essentially performed as described previously ( 20 ) with the exception that for the experiments shown in panels 5 and 6 of Fig. 4 A a mixture of affinity-purified SIT (5 μg/ml) and CD8 (AICD8.1, IgG1, 10 μg/ml) Abs was used, whereas all other experiments shown in Fig. 4 were performed using 20 μg/ml of affinity-purified SIT antibody. COS cells were transfected as previously reported ( 20 ). The constructs used were as follows: wild-type Fyn (T) or Lck cloned in pSRα expression vector (provided by Dr. A. da Silva, Dana Farber Cancer Center, Boston, MA), FLAG-tagged Syk cloned into the p5C7 vector (donated by Dr. W. Kolanus, Gene Center, Munich, Germany), and myc-tagged ZAP70 inserted into the pcDNA3 vector (a gift from Dr. R. Abraham, Mayo Clinic, Rochester, MN). Jurkat cells or J.HM1.2.2 cells were transfected as previously described ( 20 ), using 30 μg of the indicated cDNA construct together with 10 μg of the NF-AT luciferase reporter construct. At 18 h after transfection, 8 × 10 4 cells/ well were stimulated in duplicates with medium, PHA (1 μg/ml), PMA (10 −8 M) plus ionomycin (1 μg/ml), immobilized anti-TCR mAb C305, or carbachol (500 μM, Sigma Chemical Co. ) in a 96-well plate (U96 Maxisorp, Nunc immunoplate). After 6 h of stimulation cells were harvested, washed twice in PBS, and lysed for 15 min at room temperature. After centrifugation, 30 μl of the supernatants were analyzed as duplicates for luciferase activity using a luminometer . Luciferase activity is expressed as percentage of the maximal promoter activity induced by incubating the transfected cells with PMA plus ionomycin. We have recently reported the molecular cloning of a novel disulfide-linked transmembrane adaptor protein, called TRIM, that associates with the TCR–CD3–ζ complex in T lymphocytes ( 20 ). Four proteins were copurified with TRIM, namely three caseins as well as an additional protein that could not be identified by searching comprehensive databases using a tryptic peptide sequence tag ( 23 ). Therefore de novo sequencing of peptides was performed after esterification of the original tryptic digest as previously described ( 24 , 26 ). The fragmentation spectrum of peptide T 1 yielded complete aa sequences, whereas those of peptide T 2 and T 3 yielded only partial aa sequences. Searching EST databases with the sequence YSEVV(L/I)DSEPK determined for peptide T 1 resulted in the identification of an EST coding for this peptide as well as for additional 73 aa that all belonged to an as yet undescribed polypeptide. Reverse and forward oligonucleotides deduced from the EST sequence were used in 5′ and 3′ RACE experiments, respectively, to amplify the rest of the corresponding gene. An open reading frame of 588 nucleotides (data not shown) codes for a protein of 196 aa . The first 22 NH 2 -terminal residues of the newly identified protein are hydrophobic, suggesting that they could represent a leader peptide required for transmembrane transport. A putative extracellular domain comprising 18 aa contains one potential site for N-linked glycosylation (N 26 ) as well as one cysteine residue (C 27 ), which could be involved in the formation of an interchain disulfide bond. Residues 41–60 are highly hydrophobic and probably represent a transmembrane domain. The putative cytoplasmic portion of the protein (residues 61–196) contains several potential phosphorylation sites for protein kinase C (T 98 ) and/or for casein kinase II (S 83 and S 182 ). Importantly, it also contains six tyrosine residues, five of which could be involved in SH2 domain–mediated protein–protein interactions after phosphorylation. Two of these tyrosine residues represent YxxL/V-motifs (Y 127 and Y 169 ), indicating the possibility of a phosphorylation- dependent interaction with SH2 domains of src PTKs. In addition, tyrosines Y 90 and Y 188 are potential binding sites for the SH2 domain of the adaptor molecule Grb2. Finally, the cytoplasmic portion of the novel molecule possesses one potential ITIM (VxY 148 xxV), which might mediate interactions with the SH2 domains of cytoplasmic tyrosine phosphatases like SHP1 and SHP2 or with the inositol phosphatase SHIP ( 35 , 36 ). It is noteworthy to mention that tyrosine-based sequence motifs with similarity to the EEVPLY 90 GNL and the MCY 127 TSL motif are also present in the cytoplasmic portion of TRIM (EDTPIY 63 GNL motif and MCY 110 ASL motif, respectively). This could indicate that both polypeptides partially interact with the same proteins in T lymphocytes. In addition, similar to TRIM, the transmembrane region of the novel protein contains the sequence motif WGxxxxxG, which shows some similarity to a recently described dimerization motif in α-helices ( 37 ). Comparison of the predicted aa sequence of the new protein (preliminarily termed gp30/40) with all available databases did not reveal a significant homology with any known polypeptides (with the exception of TRIM, see above). The mouse homologue of gp30/40 was identified by using a mouse EST obtained from an EST database for screening a genomic library. The sequence information from positive clones was then used to generate the complete mouse cDNA by PCR. A comparison between the mouse and human aa sequences revealed that all five tyrosine-based signaling motifs as well as the putative dimerization motif in the transmembrane domains are conserved between the two species. However, in contrast to the human protein, mouse gp30/40 lacks a leader peptide. Previous data suggested that gp30/40, like TRIM, represents a disulfide-linked dimer that associates with the TCR–CD3–ζ complex under mild detergent conditions ( 20 , 33 ). To further characterize gp30/40, HPB-ALL cells were lysed in Brij58 containing lysis buffer to preserve weak protein–protein interactions. Postnuclear lysates were subjected to CD3 immunoprecipitation followed by in vitro kinase assay. 10% of the in vitro–labeled immunoprecipitate was directly loaded onto two-dimensional IEF/ SDS-PAGE, and the remaining 90% were incubated with lysis buffer containing 1% Triton X-100 plus 0.5% SDS in order to release the in vitro–labeled proteins from the primary immunoprecipitate ( 33 ). The released material was then subjected to reprecipitation using a polyclonal anti-gp30/40 antibody which was raised in rabbits immunized with a KLH-coupled peptide corresponding to aa 96–114. The two-dimensional IEF/SDS-PAGE analysis of the secondary immunoprecipitate shown in Fig. 2 A, panel 4, indicates that the anti-gp30/40 antibody reprecipitates an in vitro–labeled protein from the primary CD3 immunoprecipitate that produces at least four vertical stripes with mol wt between 30 and 50 kD and differential isoelectric points in IEF. These biochemical characteristics indicate that in vitro–labeled gp30/40 is differentially phosphorylated on several distinct phosphorylation sites and that its extracellular domain is highly glycosylated. Indeed, the deglycosylation experiment shown in Fig. 2 C demonstrates that endoglycosidase treatment of gp30/40 reduces its apparent mol wt in SDS-PAGE from ∼40 to ∼20 kD. Thus, glycosylation of the single asparagine residue in the extracellular portion of gp30/40 accounts for ∼20 kD of mol wt. Fig. 2 B further demonstrates that gp30/40 represents a disulfide-linked dimer as judged from its differential migration on nonreducing versus reducing SDS-PAGE. The data shown in Fig. 2 , panels 1 and 2, explain why we have not recognized the existence of gp30/40 as a part of the CD3-associated signaling complex in our previous studies. Thus, on a standard 1-h exposure of the in vitro– labeled CD3 immunoprecipitate gp30/40 is not visible, perhaps because of its migration in IEF/SDS-PAGE as a diffuse and only weakly focusing polypeptide. Only upon overexposure of the dried gels do the long stripes corresponding to in vitro–labeled gp30/40 become visible . These properties of gp30/40 also provide an explanation for the difficulties to analyze gp30/40 biochemically (see below). Expression of the gp30/ 40 gene was determined by Northern blot analysis using a gp30/40 cDNA probe. The multiple tissue Northern blots shown in Fig. 3 demonstrate that gp30/40 is strongly expressed in thymus and to a lesser extent in spleen and lymph nodes. Upon overexposure of the shown blots, weak gp30/ 40 mRNA signals are also detectable in peripheral blood leukocytes and bone marrow (data not shown), indicating that low amounts of gp30/40 are expressed not only in T cells but also in other cells of hematopoietic origin. Indeed, further analysis revealed that gp30/40 is expressed in the HPB-ALL and Jurkat T cell lines and to a lower extent in the B cell lines LAZ509 and SKW6.9, whereas it is not detectable in monocytic cell lines HL-60 and U937, nor the erythroleukemic cell line K562 (data not shown). These data collectively suggest that gp30/40 is selectively expressed in lymphocytes. The aa sequence of gp30/40 strongly suggested that it represents an integral membrane protein. We attempted to assess the subcellular localization of gp30/40 in the T cell lines HPB-ALL and Jurkat (both expressing endogenous gp30/40) by indirect immunofluorescence using an affinity-purified gp30/40 antiserum. However, our attempts to localize endogenous gp30/40 within the two cell lines remained unsuccessful, perhaps because the amounts gp30/40 that are constitutively expressed are too low to be detected by our antiserum . To circumvent this problem, we transiently overexpressed gp30/40 in Jurkat T cells and COS cells. The transfectants were then permeabilized with acetone and stained with affinity-purified gp30/40 antiserum. The specificity of the antiserum was proven in a Jurkat variant stably expressing a chimeric gp30/40 molecule in which the cytoplasmic part of gp30/40 was fused to the extracellular and transmembrane domains of CD8 . The confocal laser scan images shown in Fig. 4 A, panels 1, 2 (Jurkat cells), and 4 (COS cells) indicated that, as expected, gp30/40 appears to accumulate at the level of the plasma membrane. No reactivity of the antiserum was seen in nontransfected COS cells , ruling out the possibility of nonspecific reactivity of the antiserum with a membrane associated protein(s) unrelated to gp30/40. To further prove membrane association of endogenously expressed gp30/40 in nontransfected T cells, cytosolic and high salt–washed membrane fractions were prepared from pervanadate-treated Jurkat cells and subjected to gp30/40 immunoprecipitation followed by anti-PTYR Western blot analysis. As shown in the left panel of Fig. 4 B, gp30/40 is exclusively detectable in the membrane fraction, whereas no signal was obtained when the immunoprecipitate was prepared from the cytosolic fraction. The purity of the individual fractions was proven by Western blot analysis using antibodies directed at MAP kinase and the recently described transmembrane protein LPAP. MAP kinase was exclusively found in the cytosolic fraction, whereas LPAP selectively localized in the membrane preparation. Thus, gp30/40 is an integral membrane protein. To finally assess whether gp30/40 is expressed at the cell surface of T lymphocytes, HPB-ALL T cells were incubated on ice for 30 min with a CD3-ε mAb of the IgG2a isotype. Cells were then washed with ice-cold buffer (to remove all unbound CD3 mAbs) and lysed in Brij58-containing buffer, which preserves the association between gp30/40 and the TCR–CD3–ζ complex . After depletion of the nuclei, the preformed immune complexes were collected using protein A–Sepharose and subjected to in vitro kinase assay. The in vitro–labeled proteins were subsequently released from the primary immunoprecipitates using SDS-containing buffer and subjected to a second round of immunoprecipitation using gp30/40 antiserum. These secondary immunoprecipitates were separated on reducing 14% SDS-PAGE and analyzed by autoradiography. As a positive control, we used antisera directed at proteins that are known to associate with the TCR on the cell surface, namely CD3-ε, the ζ chains, and TRIM. The left panel of Fig. 4 C demonstrates that gp30/40 coprecipitates with cell surface–expressed CD3-ε in the HPB-ALL T cell line. This association seems to be similar to the association that is detectable in HPB-ALL cells that are first lysed and then subjected to CD3 immunoprecipitation . Similar results were obtained when Jurkat T cells were analyzed in an identical fashion (data not shown), whereas no specific signal was detectable in a TCR α chain–deficient Jurkat variant (JRT3-T3.1) lacking cell surface expression of the TCR–CD3–ζ complex . This rules out the possibility that the externally applied CD3 mAb passed the cell membrane. Thus, the data shown in Fig. 4 collectively demonstrate that gp30/40 is an integral membrane protein that is expressed at the cell surface of T cells. To investigate whether gp30/40 becomes tyrosine phosphorylated after TCR-mediated activation of T lymphocytes, HPB-ALL cells were stimulated for various periods of time with a mixture of biotinylated CD3 and CD4 mAbs that were cross-linked on the cell surface with avidin. Subsequently, cells were lysed in NP-40–containing buffer and subjected to immunoprecipitation with anti-PTYR mAb PY72. Immunoprecipitates were separated on SDS-PAGE and analyzed by anti-gp30/40 Western blot. As shown in Fig. 5 A, low amounts of constitutively tyrosine phosphorylated gp30/40 are detectable in nonstimulated HPB-ALL cells. Perhaps more importantly, the level of phosphorylation of gp30/40 strongly increased after cross-linking of the CD3 and CD4 receptor molecules. Moreover, a dramatic increase in gp30/40 tyrosine phosphorylation was induced in HPB-ALL cells stimulated with pervanadate. Similar results as shown here were obtained when gp30/40 immunoprecipitates were analyzed by anti-PTYR Western blot (data not shown). To more directly demonstrate TCR-mediated tyrosine phosphorylation of gp30/40, we transiently transfected Jurkat T cells with the above described CCG chimera. After overnight incubation, cells were stimulated for increasing periods of time with the clonotypic anti-TCR mAb C305 or, as a positive control, with pervanadate. Subsequently, CD8 immunoprecipitates were prepared from resting or activated cells and analyzed for their PTYR content by Western blot. Fig. 5 B, top, demonstrates that the chimera becomes rapidly phosphorylated on tyrosine residues after TCR triggering with a maximum of phosphorylation being observed after five min of C305 stimulation. Reprobing of the blots with gp30/40 antiserum revealed that identical amounts of the chimera were loaded onto the individual lanes . In summary, the data shown in Fig. 5 , A and B, clearly demonstrate that gp30/40 represents a substrate for TCR-activated PTKs. To analyze which tyrosine kinase can phosphorylate gp30/40 in vivo, we transiently expressed gp30/40 in COS cells together with plasmids coding for either Fyn, Lck, ZAP70, and Syk or with a combination of Lck/Fyn and ZAP70. However, in pilot experiments in which we used a cDNA coding for wild-type gp30/40 for transfection, we realized that detection of the transfected protein by Western blotting was hardly possible because of the highly variable levels of glycosylation. Therefore, we expressed a mutant of gp30/40 in COS cells in which the glycosylation site (N 26 ) was mutated to glutamine (gp30/40-N→ Q mutant). Mutation of this residue resulted in expression of a 20-kD doublet that can be detected by our antiserum . Lysates of COS cells transiently transfected with a combination of the cDNA construct coding for the gp30/40-N→ Q mutant and the above described tyrosine kinases were analyzed by anti-PTYR Western blot. At the same time, anti-PTYR immunoprecipitates were prepared from the transfected cells and investigated for the presence of tyrosine phosphorylated gp30/40 by means of anti-gp30/40 Western blotting. The left panels of Fig. 5 B demonstrate that under these experimental conditions gp30/40 becomes tyrosine phosphorylated by Lck and to a stronger extent by Fyn, but is only very weakly tyrosine phosphorylated when the molecule is coexpressed with either ZAP70 or Syk alone. However, when Fyn and ZAP70 are concomitantly expressed, tyrosine phosphorylated gp30/40 migrates at a slightly higher apparent mol wt compared with co-expression with Fyn alone. Since identical data were obtained in three independent experiments, these findings suggest that gp30/40 represents not only a substrate for src PTKs but that it can also become phosphorylated by ZAP70. To obtain initial information on the in vivo function of gp30/ 40, we transiently transfected Jurkat T cells with increasing concentrations of a plasmid coding for a full-length gp30/ 40 molecule together with a luciferase reporter gene construct driven by a triplicated NF-AT binding site of the human IL-2 promoter ( 38 ). At 18 h of transfection, expression of transfected gp30/40 was determined by Western blotting , and cells were stimulated for an additional 6 h with either the clonotypic anti-TCR mAb C305 or the polyclonal mitogen PHA. As shown in Fig. 6 A, overexpression of SIT inhibited both PHA- and TCR-mediated induction of NF-AT activity in a dose-dependent fashion. Thus, compared with the vector control, a significant reduction of the signals was already observed with 5 μg of transfected gp30/40 cDNA, whereas transfection with 30 μg cDNA resulted in a 91% reduction of the PHA signal and a 76% reduction of the TCR-induced NF-AT activity, respectively. Similarly, transient overexpression of gp30/40 in Jurkat cells resulted in a strong inhibition of the CD2-mediated pathway of T cell activation (data not shown). To assess whether the cytoplasmic portion of gp30/40 was responsible for the inhibitory effect of gp30/40 on the TCR- and PHA-mediated pathways of T cell activation, we transiently transfected a truncated mutant of gp30/40 into Jurkat cells in which a FLAG epitope was introduced at H 62 of gp30/40 followed by a STOP codon (gp30/40-STOP mutant). The Fig. 6 B, left, demonstrates that, in contrast to the wild-type protein, expression of the gp30/ 40-STOP mutant exerts no negative regulatory effect on NF-AT activity. This indicates that the negative regulatory role of wild-type gp30/40 on TCR- and PHA-induced transcriptional activity of NF-AT requires an intact cytoplasmic tail. Moreover, the finding that stimulation of the transfectants with a combination of PMA plus ionomycin bypassed gp30/40-mediated inhibition of NF-AT activation strongly suggests that gp30/40 probably regulates a proximal signaling event(s) of human T cell activation (see below). Inhibition of TCR- and PHA-mediated induction of NF-AT activity was also observed when the above described CCG chimera was expressed in Jurkat cells, whereas expression of a truncated version in which S 71 of gp30/40 was mutated to a STOP-codon (CC-STOP chimera), or of a control chimera (consisting of the extracellular domain of human HLA-A2 fused to a full length TRIM molecule, Ch_F), did not affect signaling . Collectively, these data indicate that gp30/40, when overexpressed in Jurkat T cells, acts as a negative regulator for both TCR- and PHA-mediated transcriptional activity of NF-AT. To more precisely localize where gp30/40 exerts its function during TCR-mediated signaling, we transfected a gp30/40 cDNA together with the NF-AT–driven reporter construct into a Jurkat variant stably expressing the human muscarinic receptor type 1 (J.HM1.2.2 cells). Stimulation of this heterologous receptor with carbachol results in activation of PLC via a G protein–coupled mechanism that does not involve PTK-mediated signaling ( 27 – 30 ). Fig. 6 D demonstrates that overexpression of gp30/40 in J.HM1.2.2 cells inhibited CD3-mediated induction of NF-AT activity to a similar extent as in wild-type Jurkat cells. Importantly, however, in the same cells gp30/40 did not influence signaling mediated via the muscarinic receptor. Thus, gp30/ 40 probably regulates TCR-mediated induction of NF-AT activity via a mechanism that is located upstream of activation of PLC. As reported above , gp30/40 possesses a putative ITIM in its cytoplasmic tail (VKY 148 SEV) ( 35 , 36 ). Given the strong negative regulatory effect on the induction of NF-AT activity after overexpression of gp30/40 or the CCG chimera, we next investigated whether gp30/40, in its tyrosine phosphorylated state, has the capacity to recruit SH2 domain–containing cytoplasmic phosphatases like SHP1, SHP2, or SHIP to the cell membrane. These phosphatases have been demonstrated to preferentially bind to ITIMs via their SH2 domains and seem to be involved in both positive and negative regulatory signaling pathways in lymphocytes ( 39 – 45 ). Jurkat cells were stimulated for increasing periods of time with either C305 mAb or PHA, washed, and lysed in NP-40– containing buffer. Subsequently, anti-gp30/40 immunoprecipitates were prepared and analyzed for coprecipitation of the above phosphatases by Western blotting. In several individual experiments we were unable to demonstrate a specific interaction of gp30/40 with SHP1 or SHIP (data not shown). However, as demonstrated in Fig. 7 A, appreciable levels of SHP2 coprecipitated with gp30/40 after TCR- (left) as well as PHA- (right) mediated activation of Jurkat cells. The specificity of interaction between gp30/40 and SHP2 was further proven in Jurkat cells treated with pervanadate. There SHP2 was found to specifically coprecipitate with gp30/40 , whereas the phosphatase failed to interact with the recently cloned transmembrane adaptor protein TRIM (which does not possess an ITIM). Conversely, as previously reported, the p85 subunit of PI3-K associated inducibly with TRIM , but not with gp30/40 (probably because gp30/40 lacks the YxxM-motif, which mediates the binding between p85 and TRIM [reference 20 ]). In summary, the data shown in Fig. 7 , A and B, indicate that gp30/40 specifically associates with SHP2 after T cell activation. Therefore, we termed the gp30/40 protein SIT (SHP2-interacting transmembrane adaptor protein). To investigate whether the interaction between SHP2 and SIT indeed involves the ITIM, we transiently expressed in Jurkat cells the above shown CCG chimera or mutants of this chimera in which either the complete intracellular portion of SIT was deleted (CC-STOP chimera) or in which the tyrosine residue of the ITIM was mutated to phenylalanine (CC-ITIM chimera). By expressing CD8/CD8/SIT chimeras instead of mutated SIT molecules, we could largely rule out the possibility that the transfected chimera forms disulfide-linked heterodimers with endogenous SIT and thus generates false positive results during our experiments. Moreover, the use of the extracellular domain of CD8 allowed us to perform immunoprecipitation experiments using a mouse CD8 mAb, thus preventing the possibility that the immunoprecipitates become contaminated with endogenously expressed wild-type SIT molecules. Fig. 7 C demonstrates that, as expected, a CD8 immunoprecipitate prepared from either nonstimulated or pervanadate-treated wild-type Jurkat cells (lacking CD8 expression) did not contain detectable levels of SHP2. However, when CD8 immunoprecipitates were prepared from cells that had been transiently transfected with the CCG chimera, large amounts of SHP2 were detectable in the precipitates obtained from pervanadate-treated cells. That coprecipitation of SHP2 by the CCG chimera requires the presence of the cytoplasmic part of SIT was evident from the observation that SIT/SHP2 interaction was completely lost when the cells were transfected with the CC-STOP chimera lacking the cytoplasmic portion of SIT. Perhaps more importantly, coprecipitation of SHP2 was also almost completely lost (>90% reduction as judged from densitometric analysis of the shown blot) when Jurkat cells were transfected with the CC-ITIM chimera. The latter finding formally proves that the interaction between SIT and SHP2 requires a functionally intact ITIM. We next investigated whether loss of interaction between SIT and SHP2 would abolish the inhibition of TCR- and/or PHA-mediated induction of NF-AT activity exerted by wild-type SIT and the CCG chimera. As shown in Fig. 7 D, overexpression of the gp30/40-ITIM mutant (top) or of the CC-ITIM chimera (bottom) downregulates both TCR- and PHA-mediated induction of NF-AT activity as observed for the wild-type protein. Thus, although expression of an intact ITIM is required for the interaction of SIT with SHP2, it does not mediate the negative regulatory effect of wild-type SIT or of the CCG chimera on TCR- and PHA-mediated induction of NF-AT activity. In this report we describe the purification, nanoelectrospray sequencing, molecular cloning, and functional characterization of a novel transmembrane adaptor molecule that we have termed SIT (SHP2-interacting transmembrane adaptor protein). As previously reported, SIT copurified with a recently cloned disulfide-linked dimer called TRIM ( 20 ). Most likely the expression of SIT in T lymphocytes was not appreciated earlier because of the generally low levels of expression of SIT in lymphocytes and its particular biochemical properties (high levels of glycosylation combined with variable levels of phosphorylation), which largely impair the detectability of the protein by biochemical standard methods . SIT represents a disulfide-linked dimer that is preferentially expressed in T and B lymphocytes. Thus, within the lymphatic system, expression of SIT seems not to be restricted to T cells as was recently reported for LAT ( 21 , 22 ) and TRIM ( 20 ). SIT also differs from LAT and TRIM by the presence of a single N-linked glycosylation site that is located directly adjacent to the cysteine residue that is responsible for dimerization. In fact, the cysteine residue is part of the glycosylation recognition sequence NxT. Deglycosylation experiments indicated that the carbohydrate moiety of SIT accounts for ∼20 kD of mol wt . Whether the carbohydrate chain represents a binding domain for an extracellular ligand is unknown at present but represents a good possibility. SIT becomes tyrosine phosphorylated after TCR-mediated activation of HPB-ALL or Jurkat cells . Coexpression experiments performed in COS cells revealed that SIT represents a substrate for src kinases, most notably Fyn . However, in contrast to TRIM, which is exclusively phosphorylated by src kinases ( 20 ), and LAT, which is probably a selective substrate for ZAP70 ( 22 ), SIT phosphorylation seems to be mediated by both src and syk PTKs. Thus, when ZAP70 and Fyn are coexpressed in COS cells, SIT reproducibly migrates at a slightly higher apparent mol wt than when expressed with Fyn alone. These findings could indicate that SIT phosphorylation occurs in two subsequent steps that are mediated by src and syk PTKs, respectively. One attractive model would be that, immediately after triggering of the TCR, SIT becomes phosphorylated by an src PTK. This phosphorylation event could induce a conformational change of SIT, resulting in accessibility of another tyrosine residue representing a phosphorylation site for ZAP70. To investigate this possibility it will be necessary to generate variants of the gp30/40-N→ Q mutant in which individual tyrosine residues are mutated and to investigate whether coexpression of Fyn and ZAP70 still produces a higher mol wt form of the molecule. However, consistent with a two-step model for SIT tyrosine phosphorylation is our observation that the apparent mol wt of tyrosine phosphorylated wild-type SIT (although difficult to assess because of its heavy glycosylation) also increases after prolonged stimulation of Jurkat and HPB-ALL cells (our unpublished data). Overexpression studies performed in Jurkat cells with the intention to elucidate the function of SIT indicated that wild-type SIT and the CCG chimera strongly downregulated both TCR- and PHA-mediated activation of NF-AT . Inhibition of NF-AT activity required the presence of the cytoplasmic portion of SIT as judged from analysis of Jurkat cells transfected with plasmids coding for versions of SIT lacking the cytoplasmic domain . Perhaps more importantly, the observation that the inhibitory effect of SIT could be bypassed with a combination of PMA and ionomycin strongly suggested that SIT controls an early step of T cell activation. This assumption was confirmed in a Jurkat variant that coexpresses the human muscarinic receptor type 1 and the TCR–CD3 complex. Both types of receptors activate PLC via distinct mechanisms. Thus, the TCR couples to PLC via the tyrosine kinase pathway. In contrast, the muscarinic receptor activates PLC via a pathway involving a heterotrimeric G protein. Our finding that overexpression of SIT alters signaling via the TCR but not via the muscarinic receptor thus indicates that SIT exerts its functional effect upstream of PLC activation and that it selectively regulates signaling via the PTK pathway. However, further investigations are required to elucidate which of the most proximal steps of TCR-mediated T cell activation are controlled by SIT. One major candidate for mediating the inhibitory function of SIT was tyrosine 148 which is a component of a putative ITIM. ITIMs are known to mediate noncovalent interactions with SH2-domain containing phosphatases such as SHP1, SHP2 or SHIP ( 35 , 36 , 39 – 45 ). Indeed, coprecipitation experiments demonstrated that SIT specifically interacts with SHP2 after TCR- and PHA-mediated activation of Jurkat cells . The association between SHP2 and SIT was dependent on phosphorylation of Y 148 as judged from a transient expression experiment which revealed that a SIT mutant in which Y 148 was mutated to phenylalanine (CC-ITIM mutant) had largely lost its ability to recruit SHP2 . The residual binding of SHP2 to the CC-ITIM chimera (less than 10%) might indicate that besides Y 148 a second tyrosine residue of SIT is involved in the interaction between the two molecules and that SHP2 uses both SH2-domains to bind to SIT. Such a mode of interaction would be in line with recently reported data describing the crystal structure of SHP2 ( 46 ). Experiments are underway to prove this possibility. The finding that SIT selectively associates with SHP2 in Jurkat cells might be surprising in light of previous data indicating that the binding motifs for the SH2-domains of SHP1, SHP2 and SHIP are quite similar. Indeed, coprecipitation experiments performed on pervanadate treated Jurkat cells revealed that tyrosine phosphorylated SIT strongly binds to the isolated SH2-domains of all three phosphatases in vitro (our unpublished observations). The selective in vivo association of SHP2 and SIT could thus result from a selective colocalization of both molecules within the cell. Another possibility would be that the presence of the positively charged lysine residue in the −1 position relative to Y 148 selects for a preferential association between SIT and SHP2. Importantly, despite that fact that the ITIM of SIT recruits SHP2 and that mutation of Y 148 in the ITIM almost completely ablated SHP2 binding, overexpression of a SIT-ITIM or a CC-ITIM mutant still resulted in inhibition of TCR- and PHA-mediated induction of NF-AT activity that was indistinguishable from the functional effect exerted by the wild-type protein . These data collectively suggest that, although expression and tyrosine phosphorylation of the ITIM of SIT is required to mediate its association with SHP2, the binding of SHP2 to SIT per se is not responsible for the inhibitory capacity of the protein under the experimental conditions used in this study. The finding that inhibition of binding of SHP2 to SIT does not rescue NF-AT activation could suggest that SIT serves several functions during T cell activation. One function could be the regulation of the transcriptional activity of NF-AT. In this regard SIT could serve as a gate-keeping molecule that helps to arrest nonprimed T lymphocytes in a quiescent state. Which part of the cytoplasmic domain of SIT is involved in this potential function requires further investigation. Preliminary data indicate that a mutant of the CCG chimera lacking the 50 COOH-terminal aa of the cytoplasmic domain no longer interferes with TCR-mediated induction of NF-AT activity (our unpublished observation). This strongly suggests that the 50 COOH-terminal aa of SIT are mainly responsible for its functional effect (at least with regard to the induction of NF-AT). However, the elucidation of the question, which of the 50 COOH-terminal aa are responsible for inhibition of NF-AT activity, requires establishing additional truncation mutants of SIT or SIT mutants in which the two COOH-terminal tyrosine-based signaling motifs (the Y 169 ASV motif and the Y 188 ANS motif, see below) would be individually or concomitantly mutated. Experiments addressing these questions are being performed. Additional roles for SIT (distinct from control of NF-AT activity) could emerge from its interaction with SHP2. Since it has been proposed that SHP2 is involved in both negative and positive regulatory pathways of T cell activation, further investigations are required to elucidate the consequences of SHP2-binding to SIT on T cell activation. It is important to note that further functions for SIT could result from the existence of the four tyrosine-based signaling motifs in its cytoplasmic domain that are unrelated to ITIMs and also do not represent ITAMs . Although these motifs are potential binding sites for the SH2 domain of Grb2 (Y 90 GNL and Y 188 ANS) and for src kinases (Y 127 TSL and Y 169 ASV), thus far we have not been able to establish a direct interaction between SIT and these signal-transducing polypeptides. Nevertheless, it is tempting to speculate that the four tyrosine-based binding motifs in the cytoplasmic domain of SIT contribute to its function in vivo. The three transmembrane adaptor proteins (LAT, TRIM and SIT) identified thus far altogether possess 22 potential tyrosine-based signaling motifs that probably mediate tyrosine phosphorylation–dependent interactions with SH2 domain– containing intracellular signaling molecules. The presence of these multiple tyrosine-based signaling motifs close to the membrane not only offers an explanation how intracellular signal transducers (e.g., PLCγ1, Grb2, PI3-K, SHP2, etc.) are recruited to the plasma membrane but also provides the T cell with potent tools for fine tuning T cell activation depending on the quality, quantity, and length of TCR occupancy. In addition, transmembrane adaptor molecules could also serve to integrate signaling events mediated via secondary signaling receptors. | Study | biomedical | en | 0.999995 |
10209037 | MBP was prepared from guinea pig spinal cords (Keystone Biologicals) as previously described ( 14 ). C57BL/6 (B6, H-2 b ), B6Smn.MRL- Fas lpr (B6. lpr , H-2 b ), B6Smn.C3H- Fasl gld (B6. gld , H-2 b ), and B10.PL (H-2 u ) mice were obtained from The Jackson Laboratory or produced in our own breeding colony from breeding stock obtained from The Jackson Laboratory . The congenic B10.PL. Fas lpr and B10.PL. Fasl gld mice were produced in our facility by backcrossing the mutations from the C57BL/6 background (B6. lpr or B6. gld ) onto B10.PL for six generations. Because C57BL/10 and C57BL/6 are closely related substrains ( 15 , 16 ) and neither mutation is linked to the MHC locus (MHC = chromosome 17, lpr = chromosome 19, gld = chromosome 1), transfer of the mutations from congenic C57BL/6 animals to C57BL/10.PL produces essentially congenic mutations on the B10.PL background. Mutant animals backcrossed one to six generations have behaved similarly in our active induction experiments (i.e., developed ameliorated clinical signs of EAE compared with B10.PL mice), indicating that the B10.PL. Fas lpr and B10.PL. Fasl gld mice are essentially congenic with B10.PL animals. MBP-specific TCR transgenic mice on a B10.PL background (H-2 u ; MBP-1; provided by Hugh McDevitt, Stanford University, CA) ( 17 ) were crossed with B10.PL. Fas lpr and B10.PL. Fasl gld animals to produce MBP-specific TCR transgenic mice homozygous for the lpr or gld allele. Animals were screened for the MBP-specific TCR transgene by dual labeling PBLs with 10 μg/ml FITC-conjugated anti-CD4 (H129.19; PharMingen ) and biotinylated anti-Vβ8.1,8.2 (MR5-2; PharMingen ). PE-conjugated streptavidin (Southern Biotechnology Associates) was used as secondary reagent. Unless indicated otherwise, all experiments were begun with animals that were 6–8 wk of age and, in most instances, were completed before they had reached 12 wk of age. Animals were housed under specific pathogen–free conditions at the Washington University School of Medicine facility according to Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Draining lymph nodes and spleens were removed from donor mice 10–12 d after subcutaneous immunization with 400 μg gpMBP and 60 μg Mycobacterium tuberculosis (H37RA; Difco Labs.) in IFA ( Calbiochem ). Single cell suspensions were treated with red blood cell lysis buffer ( Sigma Chemical Co. ) and washed, and 4–5 × 10 6 cells/ml were cultured with 20–25 μg/ml gpMBP in 6-well plates for 4 d. On day 4, cells were harvested and resuspended in HBSS. 5–8 × 10 7 viable cells were injected intravenously into 8-wk-old recipients (H-2 u ) that had been sublethally irradiated (350 rads) 3–6 h previously. Recipient mice were also administered 200 ng pertussis toxin (List Biologicals) intravenously on days 0, 3, and 7. Cells isolated from donor mice immunized with CFA alone were not viable after 4 d in culture with gpMBP and therefore were not transferred. Instead, control mice were sublethally irradiated and administered all three doses of pertussis toxin. For the transfer of MBP-specific TCR transgenic cells, lymph nodes and spleens were removed from transgenic mice and single cell suspensions were depleted of red blood cells and cultured for 2 d in 50 ml of medium containing 10 μg/ml gpMBP in a T75 flask. On the second day, one-half of the medium was removed, replaced with fresh medium plus antigen, and incubated for an additional 2 d. Cells were harvested and resuspended in HBSS. 10 7 viable cells were injected intravenously into 6-8 wk old recipients (H-2 u ) that had been sublethally irradiated (450R) 3-6 h previously. Pertussis toxin was not administered. Clinical signs of disease were monitored daily and were graded as follows: 0, normal; 0.5, partial loss of tail tonicity, assessed by inability to curl distal end of tail; 1, complete loss of tail tonicity; 2, mild to moderate hindlimb weakness or ataxia; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, moribund. Moribund animals were killed. Aliquots of the cells transferred were stained with 10 μg/ml FITC-conjugated anti-CD4, anti-Lyt-2 ( Becton Dickinson ) or anti-CD19 (1D3; PharMingen ), purified anti-CD11b (M1/70; PharMingen ), and/ or biotinylated anti-Vβ8.1, 8.2. FITC-conjugated goat anti–rat (1:40), and PE-conjugated streptavidin (1:300) were used as secondary reagents (Southern Biotechnology Associates). CNS tissues removed from mice perfused with buffered 2.5% glutaraldehyde were postfixed in 1% osmium tetroxide (Electron Microscopy Sciences), dehydrated through graded alcohols and embedded in EMBED 812 (EMS) as previously described ( 18 ). 1 μm sections taken from each level of the CNS (optic nerves, cerebrum, cerebellum, brainstem, and cervical, thoracic, lumbar, and sacral spinal cord regions) were placed on glass slides, stained with toluidine blue, and assessed blindly using a published scoring system from 0 to 5 for inflammation, demyelination, and axonal necrosis ( 19 ). Splenocytes from B6 mice were cultured for 5 d with an equal number of irradiated B10.PL splenocytes in 50 ml of medium in a T75 flask (∼3–6 × 10 8 B6 splenocytes/flask). On day 5, viable cells were recovered by Ficoll-Histopaque-1077 ( Sigma Chemical Co. ) density centrifugation, washed twice, and resuspended in HBSS. 5 × 10 7 viable cells were injected intravenously into 8-wk-old B10.PL or B10.PL. Fas lpr recipients that had been sublethally irradiated (350 rads) 3–6 h previously. Control mice were sublethally irradiated, but were not given B6 anti-B10.PL cells. On days 1, 3, and 5 after transfer, control and injected animals were killed for analysis. Tissue samples from spleen, liver, lung, and small intestine were removed from mice and embedded in OCT (Miles, Inc.) for immunohistochemical analysis. 9-μM frozen sections were placed on Superfrost Plus slides ( Fisher Scientific Co. ) and fixed in acetone for 10 min. Sections were stained with the appropriate Vectastain ABC-Elite kit (Vector Labs, Inc.) according to the manufacturer's instructions. In brief, sections were blocked with normal serum for 30 min and then incubated with primary antibody at 4°C overnight. Sections were washed twice and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30 min. Slides were then incubated with diluted ABC reagent for 30 min. Peroxidase reaction was developed using the VIP Vectastain substrate kit. Antibodies and dilutions used were biotinylated anti-K b (1:50; PharMingen ) and biotinylated anti-L d (30-5-7) (1:100; provided by Dr. Ted Hansen, Washington University, St. Louis, MO). Mice were anesthetized with methoxyfluorane and perfused with saline. CNS tissues were removed and embedded in OCT. 9-μM frozen sections were placed on Superfrost Plus slides, fixed in 1% paraformaldehyde for 5 min, and permeabilized in 100% ethanol for 5 min. TUNEL (TdT-mediated dUTP nick-end labeling) staining was conducted using a modification of a previously published protocol ( 20 ). In brief, slides were incubated with 0.3 U/μl terminal transferase ( Boehringer Mannheim ) and Cy3-labeled dUTP ( Amersham Pharmacia Biotech ) in TdT buffer (30 mM Tris HCl, pH 7.3, 140 mM sodium cacodylate, and 1 mM cobalt chloride) at 37°C for 1 h. The reaction was terminated in buffer containing 300 mM sodium chloride and 30 mM sodium citrate. After washing in PBS, sections were blocked with normal rabbit serum for 30 min and then incubated with either anti-CD4 (GK1.5; 1:1,000; Becton Dickinson ) or anti-CD11b (M1/70; 1:10,000; PharMingen ) at 4°C overnight. Slides were washed twice and incubated with biotinylated rabbit anti–rat secondary antibody (1:200; Vector Labs) for 30 min. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide in methanol for 30 min. Slides were then processed using the tyramide signal amplification (TSA)-direct kit (NEN Life Sciences) according to the manufacturer's instructions, counterstained with Hoechst 33258 (Polysciences, Inc.) and mounted in Fluoromount-G (Southern Biotechnology Associates). TUNEL + cells, CD4 + or CD11b + cells, and double-labeled cells were counted in two to four inflammatory lesions per mouse using two coded slides per lesion. For some experiments, “nests” of >10 immediately adjacent CD4 + cells were counted in four to five representative lower spinal cord cross-sections per mouse. We ( 11 ) and others ( 12 , 13 ) have previously shown that both the lpr and gld mutations ameliorate the clinical signs of EAE actively induced in H-2 u or H-2 b mice by immunization with MBP or myelin oligodendrocyte glycoprotein, respectively. We have also demonstrated that these mutations do not affect the production of MBP-specific Th1 T cells, the development of a Th1-mediated immune response in vivo, or the infiltration of inflammatory cells into the CNS ( 11 ). These data indicated that Fas and its ligand are important for the progression of clinical signs of EAE. We hypothesized that FasL + myelin-reactive lymphocytes contributed to the pathogenesis of EAE by lysing Fas- expressing targets in the CNS. However, our active induction experiments could not distinguish FasL expressed on activated, myelin-specific CD4 + T cells from that expressed on other inflammatory or resident CNS cells. Furthermore, because the mitigation of EAE was the dominant effect of the lpr and gld mutations, we were unable to determine whether a Fas-mediated lytic interaction might also be contributing to the recovery from actively induced disease. Therefore, to further examine the role of Fas and its ligand in EAE, we conducted adoptive transfer experiments of myelin-reactive wild-type, lpr , or gld lymphocytes into congenic wild-type, lpr , and gld hosts (H-2 u ). In our initial experiments, conventional (polyclonal) MBP-specific lymphocytes isolated from primed animals and cultured in MBP for 4 d were used as donor cells. Both MBP-specific cells and pertussis toxin were required for recipient animals to develop EAE. To complete the reciprocal transfers, it was necessary to introduce the mutations (especially lpr ) onto an MBP-specific TCR transgenic mouse. TCR transgenic wild-type, lpr , and gld cells from unimmunized mice responded equally well to in vitro stimulation with MBP, and more vigorously compared with the nontransgenic cells (data not shown). These cultures contained 62–78% CD4 + and 2–5% CD8 + T lymphocytes, 12–18% B cells, and <5% macrophages. In our initial experiments, conventional MBP-specific wild-type (B10.PL) cells were adoptively transferred into congenic wild-type, lpr , and gld hosts . Clinical signs of disease consistently appeared 20–30 d after transfer in recipients of all three genotypes. 88% of the wild-type animals developed clinical signs of EAE, with a peak disease severity corresponding to partial hindlimb paralysis (3.0 ± 0.91; median = 3). On the other hand, lpr recipients were quite resistant to the development of EAE, with only 12% of the animals exhibiting clinical disease. The two lpr mice that did develop clinical signs of EAE developed very mild disease corresponding to tail paralysis (grades 1 and 2; P = 0.032 versus wild-type). These data are consistent with our previous study in which the lpr mutation mitigated the development of actively induced EAE. In contrast to our previous study using an active induction paradigm, gld recipients of adoptively transferred wild-type lymphocytes were overall highly susceptible to disease, with an incidence of 75% and a mean peak disease severity similar to that observed in wild-type recipients (3.0 ± 1.1; median = 2.25). However, unlike in the wild-type recipients, in which all animals developing clinical signs of EAE progressed to clinical scores of 2–4, the severity of EAE in gld animals was highly variable. Some (9 out of 28) gld recipients developed only mild clinical signs of EAE (grade 0–1.5), similar to that observed in the lpr recipients. However, most (17 out of 28) gld recipients exhibited clinical scores of grades 2 to 4, and two mice progressed to the most severe stage of disease (grade 5). The adoptive transfer of MBP-specific TCR transgenic wild-type lymphocytes into wild-type and congenic lpr and gld hosts confirmed the results obtained when conventional wild-type lymphocytes were transferred . lpr recipients were resistant to the development of clinical signs of EAE transferred by transgenic wild-type cells. In contrast, all of the wild-type and gld animals developed clinical signs of EAE ∼2 wk after cell transfer, with peak clinical scores corresponding to moderate hindlimb weakness (wild-type, 2.1 ± 0.97, median = 2; gld , 2.4 ± 0.78, median = 2). Thus, the adoptive transfers of myelin-reactive wild-type T cells demonstrate that Fas expressed within the host (presumably in the CNS) plays an important role in the pathogenesis of EAE. Having established that lpr recipients develop only mild clinical signs of adoptively transferred EAE, we asked whether this amelioration of disease was actually due to a paucity of Fas + targets in the CNS, or if it was simply because the lpr host lymphocytes, which are reported to express elevated levels of FasL ( 21 ), were eliminating the activated, Fas + lymphocytes we transferred. We were unable to unequivocally identify the donor lymphocytes in adoptive transfer experiments described above. Therefore, 5 × 10 7 B6 anti-B10.PL cells (H-2 b ) from a 5-d MLR were injected into sublethally irradiated wild-type or lpr hosts (H-2 u ). Spleens were removed at various days after transfer and stained for L d , a marker of the H-2 u haplotype, and K b , a marker of the donor cells. Fig. 3 shows spleen sections removed from wild-type and lpr mice 3 d after transfer. Comparable numbers of K b -expressing cells were detected in the T cell zones, but not follicles, of both wild-type and lpr animals given donor cells. Spleens taken from wild-type and lpr animals that were irradiated but did not receive K b -expressing cells showed staining for L d , but not K b (data not shown). 5 d after transfer, donor cells were reduced to a similar degree in spleens of both wild-type and lpr recipients compared with day 3 levels (data not shown). Lpr recipients of conventional, nontransgenic lymphocytes were monitored for clinical signs of disease for the duration of each adoptive transfer experiment and therefore were not examined for histological signs of EAE. However, the fact that some lpr hosts did develop clinical signs of EAE suggests that the mutation does not preclude the infiltration of cells into the CNS. Lpr recipients of MBP-specific TCR transgenic wild-type lymphocytes (see below) were examined histologically at 9 and 17 d after cell transfer and found to have small infiltrates (grade 0.5–1) in the CNS (data not shown). This is consistent with our previous study in which we found Fas was not required for lymphocytes to infiltrate the CNS after active induction of EAE ( 11 ). These data indicate that the lpr hosts are not abnormally eliminating the adoptively transferred lymphocytes. Thus, the lack of EAE we have observed in lpr recipients is probably due to a role for host-derived Fas that is important for the progression of severe disease. The adoptive transfer of nontransgenic lpr lymphocytes led to the development of a lethal graft-versus-host disease (GVHD) in many of the wild-type recipient animals before the onset of clinical signs of EAE (data not shown). We reasoned that skewing the T cell repertoire to a predominantly MBP-specific CD4 + T cell population would prevent the transfer of GVHD in our adoptive transfer experiments, and thus introduced the lpr mutation onto an MBP-specific TCR transgenic mouse. Wild-type and gld recipients of transgenic lpr lymphocytes did not develop GVHD, but did develop clinical signs of EAE ∼2 wk after cell transfer. The incidence of clinical disease induced by lpr donor cells was lower (8 out of 14; 57%) than that induced by wild-type lymphocytes (14 out of 14; 100%) after transfer into wild-type recipients . In those wild-type recipients of transgenic lpr cells that developed EAE, the peak disease severity corresponded to moderate hindlimb weakness (2.3 ± 0.70; median = 2). Gld recipients were more susceptible than wild-type hosts to disease induced by transgenic lpr cells, with 92% developing clinical signs of EAE. The severity of EAE induced by the transfer of transgenic lpr cells into gld recipients was highly variable. The mean peak disease severity of gld recipients was 2.3 ± 0.87 (median = 2). We also examined the induction and progression of adoptively transferred EAE in the complete absence of Fas by transferring Fas-deficient ( lpr ) MBP-specific TCR transgenic lymphocytes into lpr mice . The incidence of disease transferred by transgenic lpr donor cells into lpr recipients was greatly reduced compared with the incidence observed in wild-type and gld recipients, with only 12% (1 out of 8) of lpr recipient mice developing EAE of an average peak clinical score of 0.12 ± 0.35 ( P = 0.020 versus wild-type; P = 7.96 × 10 −5 versus gld ). The onset of disease in the one lpr recipient that developed clinical signs of EAE occurred 24 d after cell transfer, which was slightly delayed compared with disease onset in wild-type and gld recipients of wild-type or lpr lymphocytes, and disease was mild (grade 1). Thus, Fas-deficient transgenic lpr lymphocytes were able to transfer EAE into wild-type and gld , but not lpr recipients. Furthermore, the disease that developed after the transfer of lpr cells was similar in onset and severity to that observed when wild-type lymphocytes were transferred. In these experiments, the transgenic lpr lymphocytes transferred could not have been eliminated by FasL + cells in the lpr recipients. Therefore it is highly unlikely that the lack of EAE we have observed in the lpr recipient mice is due to rejection of the donor lymphocytes. Instead, these data demonstrate that Fas expressed by the recipient animals (presumably in the CNS) is an important determinant for the pathogenesis of EAE. To determine the relative role of FasL expressed by MBP-reactive T lymphocytes in the initiation of EAE, conventional MBP-specific lymphocytes from gld mice were adoptively transferred into congenic wild-type, lpr , and gld recipients. The incidence of disease transferred with MBP-reactive gld lymphocytes was rather low, with only 22% of wild-type and 27% of gld animals developing clinical signs of EAE (Table I ). In the few wild-type and gld recipients that exhibited clinical signs of EAE, the peak disease severity corresponded to complete tail paralysis (grades 1, 1.5, 2, 2) and partial to complete hindlimb paralysis (grades 2.5, 3.5, 4, 4) for wild-type and gld recipients, respectively. None of the 11 lpr mice that received gld lymphocytes developed clinical signs of disease over the duration of the experiments. These data suggest that FasL expressed on the donor lymphocyte population is important for the initiation of the inflammatory cascade of events leading to development of EAE. The apparent exacerbation of clinical disease in the four gld recipients may reflect an inability of those animals to control the expansion of inflammatory cells (see below) that have initiated the disease, presumably through a Fas/FasL-independent mechanism. Similar results were obtained when MBP-specific TCR transgenic gld lymphocytes were transferred into wild-type B10.PL and congenic lpr and gld hosts . None of the seven lpr recipients developed clinical signs of EAE. The onset of disease in wild-type and gld recipients of transgenic gld lymphocytes was slightly delayed (20 ± 6), but did not statistically differ from that seen in animals receiving FasL-expressing cells. The incidence of disease transferred with gld lymphocytes was low compared with that transferred by transgenic wild-type lymphocytes. Only 12% of wild-type and 6% of gld hosts developed EAE with average peak clinical scores of 0.44 ± 1.2 and 0.13 ± 0.52, respectively. Thus, the adoptive transfer of MBP-specific TCR transgenic gld T cells further substantiates our conclusion that FasL expressed on donor lymphocytes is important for the initiation and/or progression of EAE. While analyzing the results from the transfer of conventional wild-type lymphocytes into the various hosts, we noticed that the duration of the clinical signs of EAE after the onset of disease varied by genotype of the recipient. Wild-type recipients recovered from the acute phase of EAE, regardless of the severity of disease that had developed, with most entering complete remission within 30 d of the onset of clinical signs (average = 26 ± 12 d) . Lpr recipients also recovered from acute EAE, although the duration of disease (average = 5 ± 3 d) was much shorter compared with that of wild-type recipients and correlated with their mild disease severity rather than their genotype . Surprisingly, gld recipients fell into two groups . Those gld mice that developed mild disease (grade ≤ 2) were able to rapidly recover from the acute phase of EAE (average = 20 ± 13 d compared with 31 ± 13 d for wild-type recipients [grade ≤ 2]). In contrast, all gld recipients that reached a clinical score greater than grade 2 developed a more chronic disease, exhibiting clinical signs of EAE for well over 35 d (average = 50 ± 14 d compared with 22 ± 11 d for wild-type recipients greater than grade 2; P = 2.4 × 10 −5 ). These data suggest that there is a source of FasL in the recipient that is lacking in the gld mice and is important in the recovery from severe disease. A relationship between the severity of disease and duration of clinical signs of EAE in gld recipients similar to that observed after transfer of conventional wild-type donor cells was also evident when disease was induced with TCR transgenic cells. 82% (9 out of 11) of the wild-type recipients of transgenic wild-type cells recovered from the acute phase of EAE within 15 d of the onset of clinical signs, with the remaining 2 animals entering complete remission within 20 d of clinical onset (average = 12 ± 5 d). In contrast, 57% (12 out of 21) of the gld recipients of wild-type transgenic cells, and all of those exceeding a clinical score of grade 2, exhibited clinical signs of EAE for >15 d (average = 18 d ± 10; P = 0.040). Likewise, when TCR transgenic lpr cells were transferred, only 2 out of 8 wild-type recipients remained clinically affected for 15 d or more (durations were 16 and 23 d). In contrast, 45% of gld recipients (5 out of 11) exhibited clinical signs of EAE for 15 d or more (durations were 15, 23, 30, 45, and 46 d, with the latter two still affected at termination of experiment 56 d after cell transfer). Thus, host-derived FasL can function in the regulation of ongoing disease, possibly by eliminating the infiltrating Fas + T cells. The experiments above indicated that severely affected gld recipients were unable to recover from the acute phase of EAE as well as their wild-type counterparts. In an attempt to understand the differences between acute and chronic disease, we decided to compare CNS lesions in wild-type and gld recipients of conventional wild-type lymphocytes at both early/acute (day 23 after cell transfer) and late/chronic (day 68 after cell transfer) stages of disease . Comparable levels of inflammation (grade 4) were found in the lower spinal cords of a wild-type and gld recipient during the acute phase of disease . The infiltrates were comprised primarily of CD11b + cells, many of which were also TUNEL positive . Roughly equal numbers of CD4 + cells were detected in inflammatory lesions from acutely affected wild-type and gld mice, with ∼4% of them undergoing apoptotic cell death . The only potential difference observed between wild-type and gld lesions at this stage of disease was a more diffuse infiltration, especially of CD4 + cells, in the gld recipient . 68 d after transfer, inflammation scores had diminished somewhat in the wild-type animals that had clinically recovered from EAE and in the gld recipients that still exhibited clinical signs of disease (wild-type, grade 1–2; gld , grade 1.5–2.5) . The degree of apoptotic cell death was greatly reduced in both wild-type and gld lesions compared with that seen at the acute stage of disease (data not shown). Wild-type lesions were comprised of fewer CD11b + cells that had a distribution similar to that observed in acute lesions (data not shown). A few, scattered CD4 + cells were also present in wild-type lesions . In contrast, clusters or “nests” of CD4 + cells were observed in many of the lesions in gld spinal cords , and were often surrounded by CD11b + cells . The correlation between the presence of these T cell nests and recipient genotype was not absolute, in that CD4 + cell clusters were not found in all spinal cord sections from gld recipients and small nests were observed in some sections of wild-type spinal cord. However, there was a direct correlation between the severity (and possibly the duration) of chronic disease in affected recipient animals, regardless of their genotype, and the average number of CD4 + T cell nests (Table III ). Taken in conjunction with results from our adoptive transfer experiments, we believe that the presence of many CD4 + cells in gld recipients is indicative of an impaired ability of these FasL-deficient animals to curtail expansion of activated, Fas + lymphocytes. A combined immunohistochemical and TUNEL analysis of actively induced EAE lesions from wild-type and lpr mice has also implicated a Fas-mediated lytic mechanism for the elimination of CD4 + and CD8 + T lymphocytes (Sabelko-Downes, K.A., A.H. Cross, and J.H. Russell, manuscript in preparation). The data presented here provide conclusive evidence that a Fas-mediated lytic mechanism is involved in the pathogenesis of EAE, as FasL-deficient lymphocytes transfer attenuated disease, whereas the absence of Fas in recipient animals dramatically attenuated the development of clinical signs of EAE induced by encephalitogenic, FasL-expressing lymphocytes. The pathogenic, Fas-dependent lytic interaction presumably occurs between FasL + lymphocytes and Fas-expressing targets in the CNS. FasL-deficient gld lymphocytes could transfer EAE to some gld mice, and an lpr recipient did develop clinical disease. This, along with our previous study in which EAE was actively induced in some lpr and gld mice ( 11 ), demonstrates that although a Fas-dependent lytic mechanism is important, the pathogenesis of EAE is not solely dependent upon Fas. The reported role for Fas in diabetes ( 22 , 23 ) has recently been questioned, as the adoptive transfer of diabetogenic lymphocytes into NOD. lpr mice resulted in a partial rejection of the donor cell population ( 24 ). The authors suggested that an interaction between FasL + recipient cells and Fas + donor lymphocytes contributed to donor cell rejection by the NOD. lpr mice. We believe that this process only poses a potential problem when the lpr allele is present in animals whose genetic background predisposes them to develop accelerated lymphoproliferative and autoimmune disease. C57BL/6 and B10.PL mice do not fall into this category, as the severe lymphoproliferative disease associated with the lpr mutation does not develop until animals are ∼12–16 wk old (Sabelko-Downes, K.A. and J.H. Russell, unpublished observation). B10.PL and congenic lpr and gld donor and recipient animals used in all of our experiments were 6–8 wk old at the time of transfer. Thus, donor lymphocytes were transferred into recipient mice at least 4–6 wk before the onset of severe lymphoproliferative disease. This issue was also addressed by the experiment in which we detected comparable levels of donor cells in the spleens of wild-type and lpr recipients 3 and 5 d after cell transfer . Two additional experiments substantiate a role for Fas in the pathogenesis of EAE. First, we ( 11 ) and others ( 12 , 13 ) have previously observed a difference in the severity of actively induced EAE between young (6–8 wk old) wild-type and lpr mice on the B10.PL and C57BL/6 backgrounds, respectively. Furthermore, in our study we were able to transfer Fas-deficient lymphocytes into wild-type or lpr mice and again we found that the absence of Fas in the host mitigated the clinical signs of EAE . In this transfer the donor lpr lymphocytes could not have been eliminated by FasL + cells in the lpr recipients. It is highly unlikely that the lack of EAE we have observed in lpr recipients results from rejection of the donor lymphocyte population. Thus, the data in this paper support our conclusion that a Fas-dependent mechanism is important for the pathogenesis of EAE. Our adoptive transfer experiments have also revealed a role for a Fas-dependent lytic interaction in the recovery from disease, as most FasL-deficient gld recipients developed chronic EAE. This Fas-dependent regulatory mechanism apparently involves targets, and possibly effectors, that are distinct from those involved in the Fas-mediated lytic mechanism that helps to initiate EAE. The FasL-expressing effector cells are host-derived, and could be resident CNS cells or cells recruited into the CNS during inflammation, whereas the targets are likely to be activated, Fas + lymphocytes infiltrating the CNS. Wild-type recipients of transgenic lpr lymphocytes did not develop prolonged signs of EAE. This suggests that a population of activated lymphocytes that are targets of Fas-mediated lysis during the recovery from EAE may be host-derived cells recruited into the CNS during the course of disease. In gld recipients, these Fas + host-derived cells and any donor lpr lymphocytes present in the CNS could help establish chronic EAE. We have found that Fas-deficient CD4 + lymphocytes were able to invade the CNS and that severe inflammation developed in the CNS of FasL-expressing wild-type hosts. This indicates that FasL does not provide a primary defense against infiltration of activated T cells (i.e., immune privilege). However, our adoptive transfer experiments suggest that once FasL is induced on cells recruited into or residing within the CNS by inflammation, it may function in a mechanism analogous to that invoked for immune privilege ( 25 , 26 ) to limit the expansion of activated T cell populations in the CNS. In gld recipients, infiltration of the CNS does not induce functional FasL, and consequently lymphocytes can accumulate and continue to damage the CNS. Although Fas-dependent T cell suicide could also occur, we were unable to ascertain the relative contribution of this autonomous lytic interaction to the elimination of the infiltrating T cell population from the experiments described here. Nevertheless, any potential treatment for MS designed to disrupt Fas-mediated lytic interactions must take into consideration this dual role for FasL in EAE. This paper provides in vivo functional evidence implicating both donor lymphocytes and host-derived elements as FasL + effectors that contribute to the progression of EAE. It further suggests the host CNS is a major source of Fas-expressing targets. Although this study has not determined the specific cell types that serve as targets and effectors of Fas-mediated lytic interactions in EAE, we can speculate as to their identity. It is likely that oligodendrocytes are the Fas + targets of myelin disruption in EAE, as they have been identified as Fas-expressing cells in MS lesions ( 6 – 8 ) and are able to express Fas and serve as lytic targets in vitro ( 6 ). FasL has been detected on glial cells ( 7 ), and in particular microglia ( 6 , 8 ), in MS lesions, making these resident cells of the CNS candidate effectors of Fas-dependent lysis. Macrophages recruited into the CNS during EAE are also potential effectors of Fas-mediated death. In sum, these experiments have described both a novel pathogenic mechanism and a regulatory process of T cell– mediated autoimmune disease in the CNS, each mediated by an interaction between Fas and its ligand. We propose the following model to describe the dual role of Fas-dependent lytic interactions in the initiation of and recovery from EAE. After an initial stimulation, the activated and differentiated T cell enters the CNS, encounters its antigen presented by a resident APC (e.g., endothelial cells, microglia, astrocytes) and is activated for the second time. At this point, the Th1 cell can function as an effector and/or target of Fas-dependent lysis. The effector T cell also secretes proinflammatory cytokines, including IFN-γ and LT/TNF-α, which can enhance expression of MHC molecules and promote cytokine/chemokine production by microglia and astrocytes ( 27 – 29 ). IFN-γ and TNF-α can also induce expression of Fas ( 30 – 32 ) or FasL ( 33 , 34 ) on various cell types. Consequently, they may contribute to the pathogenesis of EAE by augmenting expression of this lytic receptor on oligodendrocytes, for example, and/or by inducing expression of its ligand on microglia or astrocytes. Although the relative contributions of the effector cell populations is not clear, it is likely that FasL expressed by infiltrating lymphocytes plays a primary role in the initiation of disease, as it seems the FasL expressed on cells residing within or recruited into the CNS must be induced by products of the inflammatory cascade. However, upon induction, FasL expressed on cells present in the CNS may function in the pathogenic process, and certainly becomes a major participant in the recovery from EAE. The balance achieved between the Fas-mediated death of CNS cell targets, which would enhance the progression of EAE, and the Fas-dependent death of infiltrating T cells, which would contribute to remission of disease, will in part determine the clinical manifestations of EAE. | Study | biomedical | en | 0.999997 |
10209038 | Blastomyces dermatitidis ATCC strains 26199 and 60915 were used for gene disruption. The wild-type, parental strain 26199 was isolated originally from a human patient and is highly virulent in an experimental mouse model of infection ( 14 ). The genetically related strain 60915 was derived after repeated passage of strain 26199 in vitro and is reported to be 10,000-fold less virulent in mice ( 15 ). The targeting vector pQWhph was constructed as follows: pQE32/WI-1 (8.3 kb) was derived from a Qiagen ® expression vector pQE32 (3.5 kb) and an AccIII fragment of the genomic WI-1 gene (4.8 kb) ( 8 ). A BamHI site in the 3′ UTR of WI-1 was removed by HinDIII digestion and religation. Another BamHI site in the 5′ UTR was removed by EcoRI–NruI deletion. The resulting plasmid, pQWΔΔ, was digested with BamHI to excise 1.4 kb of WI-1 coding sequence. A 1.4-kb hph cassette ( E. coli hph driven by 375 bp of WI-1 upstream sequence) was amplified from pWI-1P ( 13 ) using PCR primers TB No. 1 (5′-ATC GGATCC TCGAGGTTTTGGCTTAGGCTC-3′) and TB No. 2 (5′-ATC GGATCC GGTCGGCATCTACTCTA-3′), which added BamHI sites (underlined). The hph cassette was ligated into the pQWΔΔ BamHI-digested vector, which was then linearized with HinDIII, and the 1.4-kb HinDIII fragment containing the 3′-untranslated region of WI-1 was ligated back into place. The orientations of the hph cassette and the HinDIII fragment were verified by restriction analysis. pCB1528, containing the sulfonyl urea resistance gene of Magneportha grisea , was provided by Drs. James Sweigard ( Dupont ) and Paul Szaniszlo (University of Texas, Austin, TX) ( 16 ), and was used for reconstitution of WI-1 in knockout strains. B. dermatitidis was maintained in the yeast form by growth on Middlebrook 7H10 agar medium containing oleic acid–albumin complex (OADC; Sigma Chemical Co. ). Liquid cultures of yeast were grown in Histoplasma macrophage medium (HMM) 1 ( 17 ) on a rotary shaker at 200 rpm. All cultures were maintained at 37°C. To measure the growth rate of yeasts, cells were grown synchronously in HMM and inoculated at a concentration of 2 × 10 4 per ml into 50 ml of fresh medium. Cultures were incubated at 37°C on a rotary shaker at 200 rpm for 72 h. Growth rates were monitored every 24 h by both hemacytometer cell count and OD 600 . Doubling time was calculated from the results of hemacytometer cell counts according to the formula: No. of divisions = (Log Nt − Log No.)/Log 2, where Log No. is the number of yeasts at the starting point and Log Nt is the number of yeasts at each time point analyzed. The doubling time is then equal to 24 h divided by the number of divisions. Results are the mean ± SD of three replicates per time point. B. dermatitidis yeast cells of strains 26199 and 60915 were transformed with 5–10 μg of XbaI-linearized pQWhph, using electroporation conditions previously described ( 13 ). Transformants were selected on HMM agar containing 200 μg/ml of hygromycin B. Replicates plated onto nitrocellulose membranes overlaying brain–heart infusion agar (Difco Labs.) were lysed with 0.2 M NaOH, 0.1% SDS and 0.5% mercaptoethanol as previously described ( 18 ). Membranes were probed with pooled anti–WI-1 mAbs DD5-CB4, AD3-BD6, BD6-BC4, and CA5-AA3 ( 19 ) using standard immunoblotting techniques (hybridomas were provided by Drs. Errol Reiss and Christine Morrison, Centers for Disease Control, Atlanta, GA). The multinucleate nature of B. dermatitidis yeast had to be addressed to isolate cells with a WI-1–negative phenotype, as the presence of heterologous, “silent” nuclei might allow phenotypic reversion. To obtain genetic homogeneity at the WI-1 locus, each candidate isolate was taken through several rounds of single-cell isolation on selective medium with resultant colonies re-screened as above. This protocol has been shown to render transformants of multinucleate fungi homogenous for altered genes ( 20 , 21 ). Resulting candidates were screened for evidence of gene replacement by PCR. Primers internal to the WI-1 gene were used to determine if candidates contained an intact WI-1 locus. If an intact locus was not detectable, as in candidates 55 and 99 (described in Results), homologous recombination was assessed by amplifying the junction between the transformed hph gene and sequences 5′ to the WI-1 promoter (not on the transforming vector) . Primers were 5′-TTGTTTGTCTCTGCCCCGTTTTC-3′ (forward) and 5′-CGTCGCGGTGAGTTCAGGCTTTTTC-3′ (reverse). Knockouts were confirmed by Southern blot analysis as described below. To restore the expression of WI-1 in knockout strains, yeast were cotransformed with pCB1528, together with WI-1 genomic clone 1 ( 8 ). After electroporation of 10 7 yeasts with a pool of 10 μg of each plasmid, transformants were selected on HMM plates containing 150 μg/ml of chlorimuron ethyl (Chem Service). Out of 19 total transformants, 18 were found to produce WI-1 in the colony immunoblot assay noted above. Transformants producing the most WI-1 were passed serially on HMM under selection as above to obtain a genetically homogeneous isolate. After repeated passage, strain 4/55 appeared closest to the parental strain 26199 in WI-1 production and was chosen for further study. The presence of WI-1 protein was assayed by immunofluorescence staining, SDS-PAGE, and Western blotting. Immunofluorescence staining was performed as previously described ( 8 – 10 ). In brief, yeast (10 6 cells) were stained for WI-1 indirectly using 1 μg anti– WI-1 mAb DD5-CB4 followed by goat anti–mouse IgG-FITC. Stained cells were inspected for fluorescence microscopically using an Olympus BX60 fluorescent microscope or a FACScan ® flow cytometer ( Becton Dickinson ). Cell-associated proteins were extracted by boiling yeast in treatment buffer containing 1.5% SDS, and 5.0% 2-ME for 3–5 min, followed by analysis of the cell-free material by SDS-PAGE and Western blotting using anti–WI-1 mAb DD5-CB4 as previously described ( 8 ). For Southern blot analysis, chromosomal DNA was prepared by grinding cells in liquid nitrogen and extracting them in detergent as previously described ( 13 ). Purified DNA was restricted with XbaI ( Promega Corp. ) at a ratio of 40 U/10 μg of DNA, incubated at 37°C overnight, and then separated on 1% agarose gel and transferred to nitrocellulose membrane. A 100-ng aliquot of probe was labeled with α-[ 32 P]dCTP to a specific activity of 10 9 cpm/μg using random oligonucleotides as primers ( Amersham Pharmacia Biotech ). Washed Southern blots were used to expose Kodak XAR-5 film with intensifying screens at −80°C. Murine macrophage cell line J774.16 ( 22 – 25 ), provided by Dr. Arturo Casadevall (Yeshiva University, NY), was used in most in vitro binding and phagocytosis assays. Further experiments were done with resident peritoneal macrophages of BALB/c mice. Macrophages were grown in DMEM ( GIBCO BRL ) with 10% heat-inactivated fetal calf serum (HyClone Laboratories Inc.), 10% NCTC-109 medium, and 1% nonessential amino acids ( GIBCO BRL ), and plated at 2.5 × 10 4 cells per well in 16-well tissue culture Chamber Slides (Nunc Inc.). Cells were stimulated with 500 U/ml of recombinant murine IFN-γ ( Boehringer Mannheim ). After overnight incubation at 37°C/8% CO 2 , medium in each well was replaced with fresh medium containing 500 U of IFN-γ/ml, 3 μg/ml of LPS ( Sigma Chemical Co. ) and B. dermatitidis yeasts. Binding and phagocytosis of yeasts was analyzed in vitro as previously described ( 22 – 25 ). In brief, yeasts were heat-killed for 45 min at 65°C and stained with rhodamine isothiocyanate (RITC) (10 μg/ml). Assays done in the presence and absence of complement used 10% normal mouse serum (NMS) and heat- inactivated NMS, respectively. Complement was inactivated by heating NMS at 56°C for 30 min. Yeasts and macrophages were incubated at an E/T ratio of 1:4 for varying periods at 37°C/8% CO 2 . Unattached yeasts were removed by washing wells three times with PBS. Attached but uningested yeasts were stained with 0.1% Uvitex 2B (Specialty Chemicals for Medical Diagnostics, Germany) for 30 s. Cells were fixed in 1% paraformaldehyde for 15 min. After fixation, glycerol was added to the slide. To quantify binding and phagocytosis, the number of yeasts attached to and ingested by 100 macrophages was counted at ×1,000 using a U-MWU fluorescence cube in an Olympus BX60 microscope (Leeds Precision Instruments, Inc.). The association index is defined as the number of attached and ingested yeasts divided by the number of macrophages counted. The ingestion index is defined as the number of yeasts ingested per macrophage. Results are expressed as the mean ± SEM of at least three experiments. Binding of yeast to lung tissue was analyzed using modifications to an ex vivo assay described previously ( 26 ). Lung tissue from a healthy mouse was embedded in O.C.T. compound (Miles Inc.), frozen in liquid isopentane at −80°C, and sliced into thin, 6-μm sections in a cryostat at −20°C. Sections were applied to Superfast/plus ® -coated glass slides (Fisherbrand; Fisher Scientific Co. ) and air dried. RITC-stained yeasts (10 6 ) in 0.1 ml HBSS containing 20 mM Hepes, 0.25% BSA, and 3 mM CaCl 2 , were added to a wax-inscribed circle of the tissue section. Slides were incubated for 60 min at 37°C, washed three times with HBSS to remove unattached yeasts, and fixed with 1.25% glutaraldehyde for 35 min at room temperature. Binding of yeasts was enumerated by counting the number that adhered to a 0.01 mm 2 area of the slide viewed at ×600 using an Olympus IX50 fluorescent microscope (Leeds Precision Instruments). Results are expressed as the mean ± SEM of at least six experiments. Male BALB/c mice ∼5–6 wk of age (Harlan Sprague Dawley) were infected intranasally with B. dermatitidis yeasts as previously described ( 27 ). In brief, mice were anesthetized with inhaled Metafane ® (Mallinckrodt Veterinary Inc.) to administer a 25-μl suspension of yeast cells dropwise into their nares. The minimum number of yeasts needed to achieve a lethal infection was established in preliminary work as 10 2 intranasally for ATCC 26199, and 10 6 for ATCC 60915. Mice were housed according to guidelines of the University of Wisconsin Animal Care Committee, which approved this work. Kaplan Meier ( 28 ) survival curves were generated for mice that received a lethal infection. Survival times of mice that were alive by the end of the study were regarded as censored. Time data were analyzed by the log rank statistic and exact P values were computed using the statistical package Stat Xact-3 by CYTEL Software Corporation. The survival of two groups are considered to be significantly different if the two-sided P value is <0.05. When multiple comparisons were made simultaneously, P values were adjusted according to Bonferroni's correction to protect the overall significance level of 0.05. Differences between B. dermatitidis strains in the number of CFU in tissue were analyzed statistically using the Wilcoxon Rank test for nonparametric data ( 28 ). Differences between strains in binding and phagocytosis were analyzed using methods for standard analysis of variance ( 29 ). P < 0.05 was considered significant. Our gene targeting efforts capitalized on the preferred fate of incoming DNA in B. dermatitidis , which is integrative transformation ( 13 ). Substantial WI-1 DNA flanking the hph selectable marker was used to target the knockout vector pQWhph and achieve the desired crossover event . WI-1 was disrupted by allelic replacement in ATCC strains 26199 and 60915. Presumptive evidence of homologous recombination was sought by PCR amplification of the junction between the hph gene on the transforming vector and the upstream sequences 5′ to the WI-1 promoter (not on the transforming vector) . Either of two possible products could signal such an event. A joint fragment of 1.6 kb would be amplified if chromosomal DNA had recombined at the 1 kb of WI-1 flanking sequence on pQWhph, whereas a joint fragment of 675 bp would be amplified if chromosomal DNA had recombined within the WI-1 minipromoter (375 bp) that directs expression of the hph gene. In each positive candidate (strains 55 and 99) the amplified joint fragment was 675 bp . To verify the amplified product's authenticity, it was digested with EcoRV and AatII ; the resulting DNA fragments corresponded in size to predictions based on the genomic sequence of WI-1 ( 8 ). Southern blot analyses described below confirmed the initial PCR results and demonstrated allelic replacement of WI-1 after screening 60 transformants of ATCC 26199 and 100 transformants of ATCC 60915. The frequency of homologous targeting at the WI-1 locus was 1–2%. WI-1 expression was restored in one of the knockout strains, 55, by means of gene transfer involving cotransformation with pCB1528 and genomic clone 1 containing WI-1 (as described in Materials and Methods), yielding strain 4/55. The phenotype and genotype of the knockouts and the reconstituted strain were established by anti–WI-1 mAb fluorescence staining, Western blots of extracted protein, and Southern blot analysis . Surface WI-1 was not detectable on knockout strain 55 by either FACS ® analysis or Western blotting of extracted cell wall proteins . In contrast, the amount of surface WI-1 on the reconstituted strain 4/55 was comparable to, if not greater than, the amount detected on the parental strain. Similar results were observed in isogenic strains ATCC 60915 and knockout strain 99 (data not shown). To confirm that WI-1 had been disrupted by allelic replacement, and to analyze the nature of the transforming event, Southern blot analyses were performed . The upstream WI-1 probe hybridized to a 9.3-kb XbaI fragment in DNA from parental strains 26199 and 60915, but not knockout strains 55 and 99. Instead, the WI-1 probe hybridized to a novel fragment of ∼8.3 kb in each of these knockout strains. The hph gene probe also hybridized with this same 8.3-kb fragment in the knockouts, indicating that the residual, upstream sequence for WI-1 and the hph sequence were on the same DNA fragment. For each knockout strain, the presence of a single band hybridizing with the WI-1 probe, which differs in size from the respective one in the parental strain, indicates a disrupted WI-1 locus without other ectopic integrations. The size of this fragment in knockout strains , together with the size of the joint fragment amplified in PCR , suggests that transforming DNA crossed over at the 375-bp stretch of homology (minipromoter) with the 5′ region of WI-1, excising ∼1 kb of coding sequence. This event probably placed the full 5′ region and the complete, endogenous WI-1 promoter upstream of E. coli hph , perhaps transcribing hph more efficiently. Southern blot analysis of reconstituted strain 4/55 demonstrated that the WI-1 transgene was located on a single XbaI fragment that differed in size from the retained 8.3-kb fragment harboring an hph -disrupted copy of WI-1 . This indicated that the WI-1 transgene had integrated ectopically, rather than homologously, into the chromosome in a single copy. Thus, phenotypic and genotypic analyses demonstrated that isogenic strains differing in the expression of WI-1 had been created: wild-type parental strain 26199, with a high level of expression; strain 55, devoid of WI-1; and strain 4/55, with expression restored to the level of wild-type, or perhaps higher. In addition, parental strain 60915 containing WI-1 and its isogenic knockout strain (99) lacking WI-1 were also developed . Be cause WI-1 is believed to bind yeasts to macrophages ( 8 – 10 ), we investigated whether WI-1 knockout yeasts were capable of binding. Knockout yeasts bound and entered murine macrophages poorly . The number of knockout yeasts bound to or inside of macrophages in vitro was far lower than that for wild-type yeasts at all time points between 15 min and 6 h of incubation. At 6 h, the association index for the knockout yeast was one-sixth the value for the wild-type yeast, and the ingestion index was one-eighth as high. These yeast strains bound resident mouse peritoneal macrophages in a manner similar to that observed for the macrophage cell line J774.16 (data not shown). The defect in binding and entering macrophages was restored fully in the knockout when 10% serum was added as a source of complement or after WI-1 was reexpressed in strain 4/55 . These findings imply that WI-1 mediates binding and entry of yeast into macrophages in the opsonin-poor environment of the lung early in the course of infection, before tissues are inflamed and serum exudes into the alveolus. To determine whether knockout yeasts bound poorly within the noninflamed lung itself, we quantified the number of yeasts that adhered to thin sections of lung in an ex vivo binding assay. Again, the knockout bound poorly, whereas both the wild-type isolate and the WI-1 reconstituted isolate 4/55 bound avidly , emphasizing the key role of WI-1 in promoting interactions directly with constituents of the lung alveolus. We hypothesized that yeasts unable to bind lung tissues and enter macrophages during infection would be much less virulent, and tested this in a previously described murine model of lethal pulmonary blastomycosis ( 27 ). All mice infected with the wild-type strain ATCC 26199 at a dose of 10 2 yeasts died from an overwhelming pulmonary and disseminated infection several weeks after inoculation, whereas all mice infected with the same dose of isogenic, WI-1 knockout strain 55 lived and appeared healthy during observation over 72 d . Mice infected with either 10 3 or 10 4 yeasts of knockout strain 55, which are respectively 10 and 100 times the lethal dose of wild-type yeast, also survived and appeared healthy during the 72-d observation period. Finally, lethality studies comparing parental strain ATCC 60915 ( 15 ) and its isogenic WI-1 knockout strain 99 yielded similar results (data not shown). Representative mice infected with knockout strain 55 were killed 3 wk after they were infected with 10 3 yeasts. They had <600 organisms in the lungs and none in the liver or spleen, suggesting that strain 55 produces a self-limited infection. To analyze the evolution and clearance of infection with the knockout and wild-type strains over time, groups of mice were tested serially for the burden of infection 1–14 d after they were infected intranasally with 10 4 yeasts. The number of yeasts in the lungs of mice infected with the knockout remained low throughout the study interval, whereas the number for the wild-type strain 26199 grew steadily; a sharp difference in the burden of infection between the strains was evident by 3 d and became statistically significant after 1 wk of infection (Table I ). On postmortem examination, the lungs of mice infected with the knockout were macroscopically normal, but contained a small number of well-formed granulomas with sequestered organisms . The lungs of mice infected with the wild-type strain were filled with organisms and widespread inflammation. Taken together, these findings suggest that eliminating WI-1 expression greatly reduces the pathogenicity of the yeast. We found no evidence for a growth defect in strain 55 knockout yeasts, as an alternative explanation for its reduced pathogenicity. Doubling times in liquid culture were calculated to be 17.5 h for the knockout strain and 17.1 h for the wild-type strain at 48 h after inoculation into liquid, which represented the peak log phase of growth. Wild-type parental yeasts and strain 55 knockout yeast appeared morphologically similar on agar and on microscopic examination. However, knocking out WI-1 in strain 99 led to excessive elongation into pseudohyphae, with few round yeast cells like those seen in parental ATCC strain 60915 (data not shown). This could be due to accumulated defects in the cell wall, as parental strain 60915 also has diminished α-(1,3)-glucan ( 30 ). We next confirmed that loss of WI-1 is directly responsible for reduced virulence of strain 55 in vivo, by showing that restored expression of WI-1 conferred pathogenicity. The WI-1 reconstituted strain 4/55 killed 100% of infected mice, as did the wild-type strain 26199 . Strain 4/55 accelerated the time to death and thus appeared more virulent than the parental strain 26199. Strain 4/55 yeast also displayed larger amounts of WI-1 than did the parent strain, as observed in FACS ® and in Western blots of yeast cell extracts , suggesting a possible dose effect on virulence. To assess the appearance and extent of infection with strain 4/55, mice were analyzed at the time of death. These mice demonstrated severe pulmonary and disseminated disease with >10 7 yeasts in the lungs and >2.5 × 10 4 yeasts in the liver, indicating that they died from an overwhelming infection, similar to that observed with wild-type yeast. Formal proof of the importance of a virulence determinant requires fulfillment of a “molecular” Koch's postulate: loss of virulence upon gene disruption, and restoration of virulence upon gene reconstitution ( 31 ). Prior studies of systemic dimorphic fungi have implicated candidate virulence factors through correlative and other means ( 32 ), but none of the observations have been substantiated by genetic tests, leaving their validity open to debate. Past studies that have implicated WI-1 in adherence, pathogenesis, and virulence are one such example, using exclusively nongenetic methods. Genetic intractability of B. dermatitidis and related fungi has, until very recently, prevented definitive studies. In addition to the transformation system recently described for B. dermatitidis ( 13 ), high efficiency transformation can be accomplished in the dimorphic fungus H. capsulatum ( 33 ), and the molecular and genetic tools for manipulation of that pathogen are now in hand ( 33 – 35 ). In this study, we analyzed the pathogenetic role of WI-1 in B . dermatitidis by reverse genetics, overcoming past limitations in virulence studies of systemic dimorphic fungi. Here, the WI-1 locus was targeted and disrupted by allelic replacement using the dominant selectable marker encoding resistance to hygromycin B. Evidence of homologous gene targeting and elimination of WI-1 expression was provided at both the DNA and protein levels. The frequency of homologous gene targeting at the WI-1 locus ranged from 1 to 2% in the two independent strains studied, 26199 and 60915, which compares favorably with rates observed in a study of H . capsulatum . Woods et al. ( 35 ) reported that homologous gene targeting is a rare event in that pathogen, occurring at a frequency of <10 −3 at the URA5 locus. Several reasons could account for the higher frequency of targeting in the current study of B . dermatitidis , including the differences in pathogen, genetic locus, and gene targeting strategy. We suspect that the gene targeting strategy used here was a critical feature and increased the rate of homologous recombination. Our targeting plasmid allowed a crossover event to occur in either of two locations in the 5′ region, as illustrated in Fig. 1 A: a long, 1-kb stretch of WI-1 coding sequence, or a shorter 375-nucleotide stretch of WI-1 minipromoter that served to express E . coli hph in transforming DNA. The plasmid had been designed so that the long stretch of homologous sequence in the 5′ (and 3′) region might enhance the frequency of gene targeting. Surprisingly, B . dermatitidis handled the transforming DNA in an unanticipated way in both instances in which homologous recombination was observed. Crossing over in the 5′ region occurred preferentially at the shorter stretch of homologous sequence rather than at the longer stretch. Evidence for the nature of the crossover event includes the size of the joint fragment PCR-amplified from the knockout strains , and the size of the hybridizing band detected in Southern blot analysis . This type of crossover event may have had the effect of “trapping” a more active WI-1 promoter in front of the hph coding sequence. Gene targeting by “promoter trapping” has been described in mammalian embryonic stem cells ( 36 ), where the expression of selection genes was made dependent on acquisition of transcriptional start signals from host DNA, thereby enriching for homologous recombination among preponderantly random integrations. This sort of targeting strategy may have broader application to other genes and dimorphic fungi if it renders them more amenable to allelic replacement and gene disruption by enhancing low frequencies of homologous recombination. The isogenic strain pairs created here were used to investigate the role of WI-1 in the pathogenesis of blastomycosis. Prior work using cell biological approaches suggested that WI-1 promotes binding to human macrophages ( 8 – 10 ). Genetically related strains of B . dermatitidis that vary in surface expression of WI-1 showed concordant differences in binding to macrophages in vitro: Fab fragments of anti– WI-1 mAbs blocked a portion of binding by yeast to macrophages, and purified WI-1 on polystyrene microspheres greatly enhanced their binding to the macrophages. Taken together, these findings suggested an adhesive property of WI-1. They could not address whether WI-1 is necessary or simply sufficient for binding yeast to cells, and to what extent binding by yeast would be impaired or eliminated in the absence of WI-1. Our study confirms and extends prior observations by demonstrating that WI-1 is necessary for binding the yeast to macrophages in vitro, as well to lung tissue ex vivo. Wild-type yeast bound avidly, in a time- dependent manner, whereas knockout yeast were impaired profoundly in their binding, and binding was largely restored in the WI-1 reconstituted strain. In addition, cells that bound poorly did not enter macrophages, as evidenced by low ingestion indices during the course of the incubation. In addition to data generated with the J774 macrophage-like cell line and lung tissue sections, similar trends were observed with peritoneal macrophages from mice. These observations suggest that WI-1 serves as the principal ligand mediating attachment of yeast to macrophages and host tissue, and that other ligands play a lesser role. The isogenic strains also were used to address the role of WI-1 in virulence. In a prior study ( 10 ), altered WI-1 expression in genetically related wild-type and variant strains correlated with differences in virulence. Variant strains with the lowest reported virulence surprisingly had the highest level of surface WI-1 (and binding to macrophages). Thus, a possible interpretation of that study could be that WI-1 interferes with virulence of the yeast. However, those variants also had multiple coexisting defects, including impaired secretion of WI-1 and reduced or absent surface expression of the polymer α-(1,3)-glucan. The multiple defects impeded a definite conclusion about an independent role of WI-1 or any other single component, and underscored the need for reverse genetics to allow a definitive conclusion. This study tackles this issue and demonstrates firmly that WI-1 is indispensable for the pathogenicity of B . dermatitidis . Knocking out WI-1 in wild-type strain 26199 rendered these yeast unable to establish a lethal pulmonary infection, even when administered at 100 times the minimal lethal inoculum. Similar results were observed after knocking out WI-1 in a second strain, 60915, further strengthening our conclusions about the importance of WI-1. Analyses of organ load and distribution of infection showed that wild-type yeast grew steadily in the lung and increased sharply in number by 7–14 d after infection. Although the number of knockout yeast that entered the lung was comparable to that of the wild-type immediately after infection, the number of knockout yeast in the lung remained low throughout the observation period. The persistently low number was probably not due to a growth defect, since knockout and wild-type yeast demonstrated a similar doubling time in vitro. After primary lung infection, wild-type yeast also disseminated to liver and spleen, whereas knockout yeast did not disseminate. This could reflect the ability of wild-type yeast to enter macrophages and disseminate while inside them, or possibly a burden of yeast beyond the lung's capacity. On histological examination, the pulmonary alveoli of mice infected with the wild-type were obliterated with inflammation and yeast, whereas the lungs of mice that received the knockout appeared mostly normal, except for focal well-formed granulomas with small numbers of sequestered organisms. These findings suggest that cell- mediated immunity remained intact and facilitated clearance of knockout yeasts. If this is true, administration of WI-1 knockout yeasts might vaccinate against B . dermatitidis infection due to wild-type yeasts. The indispensable role of WI-1 in pathogenicity is substantiated by this study, but the mechanism(s) whereby the molecule exerts its effect requires more investigation. A deeper understanding of WI-1 and other fungal virulence mechanisms will be important in developing new pharmacological and immunological approaches to management of systemic fungal infections. Moreover, insight into where and when fungal virulence determinants are expressed in vivo and how virulence genes are regulated should provide new information about how pathogenic fungi sense the inimical environment of the host and adapt to it successfully. Our study marks a significant first advance toward these goals by establishing WI-1 as a key virulence determinant of B . dermatitidis worthy of further investigation. | Study | biomedical | en | 0.999997 |
10209039 | Protein A from S. aureus , protein G from group C Streptococcus ssp., the IgG binding fragment BB, and protein G′ were purchased from Sigma Chemical Co. ZZ was expressed and purified as previously described ( 49 ). Toxin α was purified from Naja nigricollis venom (Pasteur Institute, Paris, France). Purity of the toxin was assessed by both reverse phase HPLC and isoelectric focusing. Biorex 70 and Bio-Gel P2 were obtained from Bio-Rad Labs. N -succinimidyl-3(2-pyridyldithio) proprionate (SPDP) was purchased from Pharmacia , and N -succinimidyl 4-( N -maleimidomethyl) cyclohexane-1-carboxylate (SMCC) was purchased from Pierce Chemical Co. N -hydroxysuccinimidyl-fluorescein-5(6)-carboxamidocaproate (NHS–FCC) was obtained from Sigma Chemical Co. The HPLC C18 column (25 cm × 10 mm) was from Vydac. All solvents were obtained from Merck and used without further purification. mAb B220, Mac-2, anti-CD4, and anti-CD8 were from the American Type Culture Collection. Streptavidin-PE (SAPE) was purchased from Caltag Labs. SpA was dialyzed overnight at 4°C against PBS before its modification with NHS–FCC. 52 nmol of SpA was subsequently incubated for 1 h at room temperature with 10× excess NHS–FCC. The reaction mixture was filtered through a Sephadex G15 column (26 × 1.5 cm) equilibrated in 50 mM phosphate buffer, pH 7.2. The protein that eluted in the void volume was used without further purification. 73.5 nmol of toxin α was modified with 735 nmol of NHS–FCC according to the same protocol. The procedure used for the biotinylation of protein G was similar to that described for NHS–FCC. For the covalent coupling of toxin α to MST2, we used a modified procedure of the protocol previously described for coupling of toxin α to peroxidase ( 42 ). 200 μl of SPDP (17 μM) in absolute ethanol was added to 3.38 μmol of toxin α in 1.8 ml of 0.1 M phosphate buffer, pH 7.5, containing 0.1 M NaCl. The mixture was stirred for 30 min at room temperature. The reaction mixture was filtered through a Bio-Gel P2 column (26 × 1.5 cm) equilibrated in 10% acetic acid. The protein which eluted in the void volume was freeze dried. The modified protein was dissolved in 1 ml of 0.05 M ammonium acetate, pH 7.1, and applied onto a Bio-Rex 70 column (70 × 2.5 cm) equilibrated in the same buffer. The toxin was eluted with a linear gradient of 0.05– 0.4 M ammonium acetate, pH 7.1. Fractions containing monomodified derivatives were roughly identified by analogy with the elution profile previously obtained with monoacetylated derivatives. The modified amino groups were unambiguously identified by determination of the affinities of each derivative for two toxin-specific mAbs. Each derivative was rechromatographed on a C18 10-mm Vydac column equilibrated in water containing 0.1% TFA. The derivatives were eluted by using acetonitrile as secondary solvent. Elution was over a linear gradient of 0–50% acetonitrile. Each derivative that was freeze dried had incorporated a 2-pyridyl-dithiopropionate moiety on a single lysine. 5.5 μl of dimethylformamide containing 83 nmol of SMCC was added to 8.3 nmol of MST2 dissolved in 312 μl of 0.1 M phosphate buffer, pH 7, containing 0.1 M NaCl. The mixture was left for 1 h with stirring at room temperature. The reaction mixture was then filtered through a Sephadex G15 column (20 × 0.5 cm) equilibrated in 0.1 M phosphate buffer, pH 6, containing 0.1 M NaCl. The coupling reaction was performed in three steps. (i) The extra disulfide bond at Lys 27 was reduced. 8.3 nmol of the derivative was dissolved in 200 μl of 0.1 M acetate buffer, pH 4.5, containing 0.1 M NaCl and 25 mM dithiothreitol. The mixture was left under stirring at room temperature for 20 min, and the solution was filtered through a Sephadex G15 column (20 × 0.5 cm) equilibrated in 0.1 M phosphate buffer, pH 6.1, containing 0.1 M NaCl. The monothiolated toxin eluted in the void volume. (ii) (27-Nε mono thio-propionyl-lysine)– toxin α (8.3 nmol) was reacted with maleimido MST2 (8.3 nmol) overnight at 4°C. Uncoupled toxin α was removed by affinity chromatography on a Sepharose–protein G column. The conjugate was then concentrated using a Centricon ® 10 and stored at −20°C with 0.1% BSA. Microtiter ELISA plates were coated overnight with either 0.3% BSA or polyclonal human (h) IgMs (1 μg/well) in 0.05 M phosphate buffer, pH 7.4. Plates were then saturated with 0.3% BSA in 0.1 M phosphate buffer, pH 7.4. Dilutions of biotinylated toxin α, Mα2-3, ZZ, and BB were performed in 0.1 M phosphate buffer, pH 7.4, containing 0.1% BSA. For the assessment of the binding of Mα2-3 to IgM-coated plates, the mAb (3 nM) was incubated overnight at 4°C in BSA-coated plates in the presence or absence of ZZ or BB (8 nM for each SpA derivative). The incubated solutions were then transferred into IgM-coated plates and left at room temperature for 4 h. The wells were then washed five times with 0.01 M phosphate buffer, pH 7.4, containing Tween 20, and a F(ab′) 2 goat anti–mouse IgG peroxidase conjugate (Immunotech) was added and incubated for 30 min. After extensive washings, 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) was added, coloration was developed for 30 min, and the resulting absorbance was measured at 414 nm. For the assessment of the binding of biotinylated toxin α to IgM-coated plates, Mα2-3 and biotinylated toxin α were incubated overnight at 4°C in BSA-coated plates in the presence or absence of ZZ or BB. The incubated solutions were then transferred into IgM-coated plates and left at room temperature for 4 h. The wells were washed, and a streptavidin peroxidase conjugate (Immunotech) was added and incubated for 30 min. ABTS was used as a substrate as described above. All experiments were performed using DCCM1 (Biological Industries) as a synthetic culture medium, in the absence of FCS. Fixed or serial dilutions of the different Ags were preincubated in microculture wells for one night at 4°C. 5 × 10 5 splenocytes per well were then added, along with 5 × 10 4 of either T1B2 or T1C9 hybridoma. Cells were cultured for 24 h at 37°C in a humidified 7% CO 2 atmosphere. For the assessment of the presenting capacity of SpA-specific cells, various amounts of splenocytes, splenocytes enriched with SpA-specific cells, or splenocytes depleted of SpA-specific cells were added to the wells in the presence of 5 × 10 4 T1B2. The presence of IL-2 in culture supernatants from T cell hybridoma and bulk culture was evaluated by determining the proliferation of an IL-2–dependent cytotoxic T cell line (CTLL) using methyl [ 3 H]TdR (5 Ci/mmol; CEA). The data are expressed in cpm. Biotinylated toxin α (0.7 μM) was incubated for one night at 4°C in the presence or absence of SpA and Mα1 (0.6 and 0.1 μM, respectively). Splenocytes (5 × 10 5 cells) were then added and incubated for 30 min at 4°C in PBS/ 0.5% BSA. Cells were washed three times and incubated for 30 min at 4°C with SAPE (6 μg/ml). After three washes, 5,000 viable cells were analyzed on a FACS ® ( Becton Dickinson ). The binding of SpAF to splenocytes was assessed by incubating 5 × 10 5 cells for 30 min at 4°C with 50 μl SpAF (0.6 μM final) in the presence or absence of 10× excess unlabeled SpA. After three washes, 5,000 viable cells were analyzed on a FACS ® . For the assessment of the binding of the two IBPs to splenocytes, biotinylated protein G (0.2 μM final) was incubated for 30 min at 4°C with 5 × 10 5 cells in the presence or absence of SpAF. Splenocytes were subsequently washed three times and incubated for 30 min at 4°C with SAPE (6 μg/ml). After three washes, 5,000 viable cells were analyzed on a FACS ® . Cells were incubated for 30 min at 4°C in the presence of SpAF. After three washings, splenocytes were filtered through nylon and sorted at 2,000 cells/s under sterile conditions on an EPICS V flow cytometer (Coulter) using a 76-μM nozzle and the 488-nm line of an argon laser set at 300 mW. PBS was used as a sheath fluid. The sorting criteria were normal forward-angle light scatter and bright fluorescein fluorescence (515–560 nm) taken on a log scale; ∼2% of cells were positive. Biotinylated toxin α (0.7 μM) and SpA (0.6 μM) were incubated for one night at 4°C in the presence or absence of Mα1 (0.1 μM). 10 5 splenocytes, SpA unbound splenocytes, or SpA-bound cell–enriched splenocytes were then added and incubated for 30 min at 4°C in PBS/0.5% BSA. Cells were washed three times, and binding to cells was analyzed as described above. To determine the type of cells bound by SpAF, SpA-bound cell–enriched splenocytes were incubated for 30 min at 4°C with mAb–B220, Mac-2, anti-CD4, and anti-CD8, respectively (0.1 μM final for each). Cells were washed three times, and binding to cells was analyzed as described above. We investigated the influence of SpA from S. aureus on presentation of an Ag–Ab complex to Ag-specific T cells. We used a snake toxin, toxin α ( 43 ), as an Ag, two toxin α–specific T cell hybridomas, called T1C9 and T1B2 ( 44 ), and two IgG2a toxin α–specific mAbs, called Mα1 ( 45 ) and Mα2-3 ( 46 ), whose epitopes are topographically unrelated to each other. Serial dilutions of toxin α were preincubated overnight at 4°C in the presence of fixed quantities of a toxin-specific mAb (Mα1 or Mα2-3) and SpA before addition of the appropriate T cells and splenocytes from BALB/c mice. Secretion of IL-2 was determined 24 h later. As shown in Fig. 1 , the capacity of toxin α to stimulate T1B2 and T1C9 in the presence of splenocytes decreased when the Ag was incubated with either Mα1 or Mα2-3 . However, the suppressing effect caused by the two toxin-specific mAbs not only vanished in the presence of soluble SpA, but the efficacy of Ag presentation dramatically increased. Fig. 1 shows that 20–100-fold lower amounts of toxin α sufficed to cause secretion of the same amounts of IL-2 when the Ag was incubated with both toxin-specific mAbs and SpA. Therefore, the presence of an Ig-binding protein can specifically enhance presentation of an Ag complexed to an Ag-specific Ab. In these experiments, we used relatively high proportions of SpA and mAbs (0.2 μM). Nevertheless, when their concentrations decreased to 4 nM and 2 nM, respectively, being thus probably closer to physiological conditions, a potent boosting effect remained observable . It is notable that, in these experiments, the initial suppressing effect of the mAb apparently disappeared . This is no surprise, as the ratio of concentrations of mAb (2 nM) to antigen (0.1 μM) was low and, as a result, the presentation associated with the free toxin dominated and overshadowed the inhibiting effect of the mAb. The same phenomenon was observed in all experiments in which the toxin concentration was higher than that of the antibody (see below). This is not the first time that an Ag presentation has been shown to be affected by mAbs. In particular, presentation of tetanus toxin was shown to increase or decrease in the presence of some specific mAbs ( 11 , 12 ). In our study, we observed only a decrease in presentation by both toxin-specific mAbs, although they bound to topographically unrelated epitopes ( 45 , 46 ). This result might be associated with the small size (6.8 kD) of the antigen, whose mAbs cover a large proportion of the toxin surface and are thus likely to interfere with presentation of the toxin T cell epitopes, located between residues 24–41 and 32–49 ( 44 ). It was previously shown that when SpA is expressed at the surface of Staphylococcus or coupled to Sepharose, it can exert a mitogenic stimulating effect ( 18 , 47 ). To rule out the possibility that the SpA-specific boosting phenomenon described above was related to such a nonspecific property, various controls were performed. First, in the absence of any mAbs, Mα1, or Mα2-3, the toxin α stimulation efficacy was unaffected by SpA . Second, in the absence of toxin α, no IL-2 stimulation was triggered by soluble SpA and Mα1 . Third, SpA did not modify Ag presentation when the Ab was an unrelated IgG2a . Fourth, no stimulation occurred when splenocytes and T1B2 were incubated separately with SpA, toxin α, and Mα1 (data not shown). Therefore, besides the necessary appropriate immune cells, three additional components were absolutely required for the boosting effect to be observable: the Ag, an Ag-specific Ab, and soluble SpA. The presentation of free toxin α to T1B2 and T1C9 is restricted to I-A d and I-E d , respectively ( 44 ). We investigated whether or not this MHC restriction was still associated with the SpA-boosted presentation of the toxin using splenocytes from D2GD mice, which express I-A d and I-E b molecules. We observed that the capacity of toxin α to stimulate T1B2 was boosted when the toxin was added concomitantly with both the mAb and SpA , whereas under the same experimental conditions, T1C9 was not stimulated . Therefore, the SpA-dependent boosting presentation of an Ag complexed to an Ag-specific Ab remains MHC restricted and is not related to the MHC-independent genetic background of BALB/c mice. What are the mechanisms that may account for an SpA-dependent boosting presentation of an Ag complexed to an Ag-specific Ab? As already mentioned, SpA possesses multiple Ig binding sites ( 23 , 24 ) and therefore could bind simultaneously to several immune complexes, thus generating an artificial concentration effect. In other words, incubation of stoichiometric concentrations of Ab and SpA could favor formation of heterogeneous multimolecular complexes, and those possessing several toxin molecules could predominantly generate the boosting effect. To examine such a possibility, we incubated overnight a constant concentration of SpA with varying dilutions of toxin α and either a fixed concentration of Mα1 or varying concentrations of Mα1 so that the ratios of SpA/Mα1 were equal to 1.5, 4.5, 13.5, 40.5, 121.5, or 364.5 for the first to the sixth dilution, respectively. Thus, in each toxin dilution, at most one molecule of the immune complex should have bound to one SpA molecule. As shown in Fig. 3 , T cell presentation evolved similarly with toxin dilution, irrespective of whether the concentrations of Mα1 were fixed or variable. Therefore, the observed boosted T cell stimulation seems to be unrelated to the number of Ab–Ag molecules associated to an SpA molecule. Another explanation that may account for the SpA- enhanced presentation of an Ag is that SpA targets Ag–Ab complexes to APCs that possess SpA receptors on their surfaces. To investigate this possibility, we performed a series of FACS ® analyses. At first, we examined the direct binding of SpA to splenocytes using SpA labeled with FCC (SpAF). As shown in Fig. 4 , E–G, labeled SpA binds specifically to a subpopulation of splenocyte cells. We then investigated the binding of the Ag on splenocytes using SpA, a toxin-specific mAb (Mα1), and the biotinylated toxin α, which was detected by SAPE. When the biotinylated toxin α was added alone, <0.5% of splenocytes were labeled . The same result was obtained when SpA or Mα1 was added separately. In sharp contrast, when the biotinylated toxin was concomitantly incubated with Mα1 and SpA, the proportion of labeled cells increased to nearly 3% . These findings support the view that splenocytes possess SpA-specific cells and that an Ag–Ab complex binds to this subpopulation. To further validate this conclusion, we sorted SpAF- labeled splenocytes (2% of the whole population) and investigated the binding of the biotinylated Ag to this population enriched in SpA-specific cells. 32% of the positively sorted splenocytes were effectively labeled with SpAF . In the absence of both SpA and Mα1, quite a small proportion of these cells were labeled with the biotinylated toxin . In contrast, when both Mα1 and SpA were present, nearly 60% of the SpAF-labeled cells were labeled with the biotinylated Ag . However, a proportion of SpAF-labeled splenocytes were not labeled with the biotinylated toxin, even when both the mAb and SpA were present in the medium . No definite explanation presently accounts for this observation. Control experiments were carried out with the population of negatively sorted cells, which contained only 0.4% of SpAF-labeled cells . Only 0.6% of the depleted splenocytes were labeled with the biotinylated Ag in the presence of the Ab and SpA , and this value did not increase to more than 2.5% when the splenocytes were unsorted . Altogether, these experiments provide direct evidence that in the presence of SpA, an Ag–Ab complex is preferentially targeted to a particular subpopulation of splenocytes that possess SpA receptors. To identify the cell types that predominantly populated splenocytes enriched in sorted SpAF-labeled cells, we used four biotinylated mAbs (mAb B220, mAb Mac 2, mAb GK1.2, and mAb H35) and SAPE. As shown in Fig. 5 G, a shift in fluorescence intensity was observed with the B cell marker, whereas no shift was observed using macrophage, CD4, and CD8 cell surface markers . Therefore, SpAF-bound cells are mainly composed of B cells. We investigated the ability of both unsorted splenocytes (S) and splenocytes enriched with (ES) or depleted of (DS) SpAF-bound cells to stimulate T1B2 in the presence of toxin α alone or in combination with both SpA and Mα1. As shown in Fig. 6 A, 10 4 splenocytes enriched with SpA receptor–possessing cells were ∼10-fold more efficient than 10 4 unsorted splenocytes at boosting toxin α presentation in the presence of Mα1 and SpA. In contrast, 10 5 unsorted splenocytes were ∼10-fold more efficient than 10 5 splenocytes depleted of SpA receptor–containing cells . The observed differences in efficiency are not related to a modification of the processing efficiency of cells treated with SpAF during the sorting experiments, as control experiments showed that splenocytes enriched with SpA receptor–containing cells were equally as efficient as unsorted splenocytes at presenting toxin α alone . Further demonstration that positively sorted cells are more efficient than unsorted cells, which in turn are more efficient than the negatively sorted splenocytes, is also shown in Fig. 6 C, where stimulation of T1B2 by the Ab–Ag complex in the presence of SpA is shown as a function of the number of cells. Therefore, the subpopulation of splenocytes possessing SpA receptors is mainly responsible for the capacity of SpA to boost the T cell stimulation of Ag–Ab complex. The above data indicated that the Ag–Ab complexes are targeted to SpA receptors present at the surface of APCs containing predominantly B cells. In other words, the boosting effect can be interpreted as resulting from the formation of complexes that consist of a SpA receptor bound to SpA, which in turn is bound to Ab–Ag complex. To investigate the possibility that the SpA receptors may be surface Igs, we proceeded as follows. First, we selected two SpA derivatives which differ in their recognition specificity toward Igs. One of them is composed of two B domains (BB), and, like SpA, recognizes the Fab region of IgMs and IgGs as well as the Fcγ part of IgG ( 48 ). The other derivative is ZZ (Z is a mutant of domain B ) and binds to the Fcγ part of IgG but very weakly to the Fab region ( 50 ). Second, we examined the binding of either a mouse mAb or biotinylated toxin α to hIgM-coated plates. As shown in Fig. 7 A (left), the mouse IgG2a mAb Mα2-3 can bind to coated hIgMs in the presence of BB but not in the presence of ZZ. Furthermore, the presence of Mα2-3 and BB is a prerequisite for the binding of biotinylated toxin α to coated hIgMs . Therefore, formation of a ternary complex, IgM–SpA derivative–IgG, or a quaternary complex, IgM–SpA derivative–IgG–Ag, is possible, provided that the derivative can bind to both IgG and IgM. In addition, the V H domain of Mα2-3 is coded by the V H germline gene J558 ( 51 ), a characteristic that precludes the Fab of this IgG to bind SpA and BB ( 35 , 36 ). As a result, this IgG can be recognized only through its Fcγ. Therefore, the complex observed in the ELISA consists of an IgM bound by its Fab to BB, which in turn binds to the Fcγ region of Mα2-3. Third, we investigated the effect of the two SpA derivatives on the labeling of splenocytes from BALB/c mice by a mouse IgG–FITC. In these unpublished experiments, we observed that 10.8% of the splenocytes were bound by the mouse IgG2a–FITC, a proportion which increased to 23.8% in the presence of BB and decreased to 6.4% in the presence of ZZ. Fourth, we investigated the capacity of BB and ZZ to boost the presentation of the Mα2-3–toxin complex using T1C9 and splenocytes. As shown in Fig. 7 B, a boosting effect is observed when the Ab–Ag complex is incubated in the presence of BB but not in the presence of ZZ. Thus, the boosting effect requires that the SpA derivative possesses both a binding site to the Fcγ region of Mα2-3 and a binding site to the Fab of Igs, strongly suggesting that the BB–Mα2-3–Ag complex is targeted to the Fab region of surface Igs of APCs. We investigated whether a bacterial IBP other than SpA could also trigger a boosting effect. Several of the above experiments were therefore repeated using protein G from Streptococcus ssp. group C ( 52 ), which contains both a Fab binding site and an Fcγ binding site ( 53 , 54 ). As shown in Fig. 8 A, protein G boosted presentation of toxin α to T1B2 only when the toxin, Mα2-3, and protein G were concomitantly added to appropriate immune cells. In contrast, the boosting effect was not observed with protein G′, a protein G derivative that binds exclusively to the Fcγ of Igs ( 55 ). Therefore, these data suggest that the boosted presentation of an Ab–Ag complex results from a targeting by protein G to the Fab region of surface Igs of APCs. This raises the question of whether SpA and protein G recognize the same APC subpopulation. To approach this question, we performed FACS ® analyses using both biotinylated protein G and SpAF. As shown in Fig. 8 , protein G (B3, top left) and SpAF (B2, bottom right) bind to approximately the same proportion of the whole splenocyte population. Furthermore, the data presented in Fig. 8 (B4) suggest that SpA and protein G may recognize different splenocyte subpopulations. However, as we observed a slight increase in the upper right area of B4, we cannot preclude that a proportion of cells recognized by SpA and protein G are the same. The view that emerges from the above data is that presentation of an Ag is boosted in the presence of an Ag-specific Ab as a result of the targeting of the complex by an IBP to APCs that contain IBP receptors. According to this scenario, both the mAb and SpA should also undergo a boosted presentation. This was demonstrated earlier to be the case for SpA ( 56 ). To provide evidence that it is also the case for the mAb, one would need mAb-specific T cells; however, they were unavailable in our laboratory. To overcome this difficulty, we used the toxin α–specific T1B2 to tentatively monitor the presentation of an mAb that was selected for its inability to recognize toxin α ( 57 ). This mAb, an IgG1 called MST2, was covalently linked to toxin α, which became, therefore, a label to probe the presentation of MST2. As shown in Fig. 9 , the MST2–toxin α couple was efficiently presented to T1B2, and its presentation was clearly boosted by SpA. As a control, no such boosting effect was observed when the free toxin α was incubated with both the uncoupled MST2 and SpA (not shown). Therefore, only the toxin that was covalently linked to the Ab underwent a boosted presentation. This effect most probably reflects a boosting effect of the Ab itself. We conclude, therefore, that the boosting presentation of an Ab–Ag complex, as previously perceived via the stimulation of the Ag, reflects an increased presentation of both the Ag and Ag-specific Ab, a finding in agreement with the proposed scenario that an immune complex may be targeted by SpA to APC containing SpA receptors. Using a snake toxin as an Ag and two toxin-specific mAbs (Mα1 and Mα2-3), we showed that two soluble bacterial IBPs, SpA and protein G, dramatically increase the Ag-specific T cell stimulation of Ag–Ab complex. These findings are in sharp contrast to the observation that, in the absence of IBP, either of the Ag-specific mAbs inhibits presentation of the Ag. A number of observations cast some light on the origin of this IBP-dependent boosting phenomenon. Thus, for the boosted presentation of the Ag to be seen, the Ag, an Ag-specific Ab, and an IBP, SpA or protein G, are absolutely required. As SpA is likely to boost presentation of a free Ab, we propose that the SpA-dependent boosted presentation of an immune complex, as perceived through presentation of its Ag, results from the binding of SpA to the Ab of the immune complex. Three lines of evidence indicate that the Ag-boosted presentation may result from binding of a ternary complex SpA–Ab–Ag to B cells containing SpA receptors. First, FACS ® analyses demonstrated that an Ag–Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes possessing SpA-specific receptors and mainly composed of B cells. Second, the boosted presentation further increased when splenocytes were enriched in cells containing SpA-specific receptors. Third, the boosting effect largely decreased when APCs were depleted of cells containing SpA-specific receptors. Altogether, these observations suggest that the two soluble IBPs can bridge an immune complex to IBP receptors present at the surface of a particular subpopulation of APCs, thus enhancing endocytosis and presentation of the Ag. For SpA to bridge an immune complex to SpA receptors, appropriate binding sites must exist on the Ag–Ab complex. It is known that SpA can interact with the Fcγ region of mouse IgGs without impairing the binding of the Ags to their paratopes ( 19 ). In addition, SpA can bind to an alternative site located within the Fab domain of some mouse Igs ( 25 , 29 ) that is coded by V H families S107 or J606 ( 35 , 36 ). The V H domain of Mα2-3, one of the two toxin-specific mAbs that triggered the boosting effect, is coded by the V H germline gene J558 ( 51 ), whose gene product is, therefore, not recognized by SpA ( 35 , 36 ). Thus, the Fcγ region of Mα2-3 and perhaps of Mα1, whose amino acid sequence is still unknown, are likely to be responsible for the interaction of the immune complex with SpA. For SpA to bridge the Ab–Ag complexes to APCs, these cells must also possess appropriate SpA receptors. Four lines of evidence suggest that these receptors are surface Igs and most probably IgMs. First, we observed that the quaternary complex IgM–SpA (or BB)–Mα2-3–Ag can occur in vitro. Second, binding of a soluble IgG to splenocytes is increased only in the presence of an IBP derivative that possesses the capacity of binding concomitantly to Fab and Fcγ moieties. Third, for the boosting effect to be observed, an IBP with the same characteristics is also strictly required. Fourth, SpA preferentially targets Ab–Ag complex to a subpopulation of B cells. Because the surface IgMs offer an alternative binding site on the Fab domain, we suggest that they constitute the most plausible SpA receptors. Other possible SpA receptors are the Igs bound in vivo to FcRs present at the surfaces of macrophages ( 58 ). Although we detected no macrophages in the subpopulation of splenocytes enriched in cells containing SpA receptor, we cannot rule out their participation in the boosting effect. Therefore, our findings are compatible with the view that SpA may link the Fcγ part of the Ab moiety of an immune complex to cell surface, SpA-specific receptor structures such as IgMs present at the surface of a subpopulation of murine splenocytes. Protein G from Streptococcus ssp. also efficiently boosts the presentation of Ag in immune complexes. Like SpA, protein G binds to the Fc region of IgGs in the Cγ2–Cγ3 interface ( 53 , 54 ), their respective epitopes partially overlapping ( 59 ). Unlike SpA, however, protein G binds to Fab regions of IgG via the CH1 domain ( 60 , 61 ) and not to IgMs ( 52 ). Therefore, if soluble protein G acts, like SpA, as an enhancer of immune complex presentation, its bridging capacity is likely to be different. Presumably, protein G predominantly focuses immune complexes to APCs carrying IgGs. Several lines of evidence, mostly taken from the literature, indicate that SpA may react similarly with both human and mouse Igs. First, hIgs bind at two independent sites on SpA ( 14 , 26 – 28 , 30 ). Second, mouse Igs S107 and J606 are related to the human V H genes of family 3 ( 62 ), and several works have shown that hIgG F(ab′) 2 and hIgM reacting with SpA derive from the V H III family but do not have markers for other families ( 32 – 34 ). More precisely, 16/26 potentially functional germline V H III genes encode SpA reactivity ( 63 , 64 ). Third, SpA–Sepharose or SpA-containing S . aureus can stimulate B cells expressing V H gene segments encoded by a set of genes belonging to the V H III family ( 47 ). Fourth, as reported above, the quaternary complex hIgM–SpA–mAb–toxin is sufficiently stable to be detected in ELISA assays. Plausibly, therefore, soluble SpA could bridge the Fc region of soluble hIgs to cell surface hIgs coded by one or more of the 16 germline V H III genes encoding SpA reactivity. Remarkably, up to 54% of human B cells are capable of binding SpA ( 64 ), whereas V H families J606 and S107 are expressed in only 10–20% of adult B lymphocytes of BALB/c mice ( 65 – 68 ) and are likely to bind SpA. Therefore, it is not impossible that the binding of SpA with hIgs could cause a boosting effect in humans similar to the one that we described here for mice. During an humoral immune response, an Ag is captured by dendritic cells, macrophages, and Ag-specific B cells, processed, and presented to Ag-specific T helper cells. This cascade of events leads to Ab secretion. The secreted Ag-specific Abs bind to the Ag, forming an heterogeneous population of immune complexes. It has been proposed that these complexes can cause a negative feedback on the specific antibody response, as a result of the diversion of Ags from their specific B cells, toward cells possessing Ig receptors, i.e., FcγR-expressing cells and RF-producing B cells ( 69 ). In this scenario, the Ag-specific helper T cells may activate B cells producing RF by intermolecular help. The data presented in this paper suggest another mechanism that might affect the immune response during an infection process by IBP-secreting bacteria. As a result of the presence of a soluble IBP, SpA for example, the Ab–Ag complexes may be targeted to B cell IgMs that can bind SpA. The SpA-binding B cells may thus present T cell epitopes from the targeted immune complexes and stimulate the corresponding T cells and may be activated by an intermolecular help mechanism. Although their specific Ags may not necessarily be present in the medium, the targeted B cells may nevertheless secrete their natural antibodies. The specificities of the produced antibodies cannot be identified a priori. However, they are likely to be large because the V H -reactive B cells are not bound by SpA via the traditional antigen binding site. It is tempting to speculate that this predicted diverting mechanism by an IBP may help the bacteria to evade the immune response. | Study | biomedical | en | 0.999998 |
10209040 | The pSRαp210BCR/ABL wild-type (WT) and the kinase-deficient K1172R, ΔSH3, ΔSH2, P1013L, and R1053L BCR/ABL mutants have been described previously ( 9 , 21 ). The ΔSH3+ΔSH2 (deletion of both the SH3 and the SH2 domains), P1013L+ΔSH2 (single aa substitution in the SH3 domain and deletion of the SH2 domain), ΔSH3+R1053L (deletion of the SH3 domain and single aa substitution in the SH2 domain), P1013L+R1053L (single aa substitutions in the SH3 and SH2 domains, respectively), p210BCR/ABL mutants were generated by replacing the EcoRI-KpnI (containing the SH3 domain) and the KpnI-BsrGI (containing the SH2 domain) fragments of BCR/ABL with the corresponding fragments containing the indicated mutation or lacking the region corresponding to the SH3 or the SH2 domain. All constructs were sequenced to confirm the presence of specific mutations. The pSRαp185 BCR/ABL triple mutant (TM) (Y177F+R522L+Y793F) and Δ176–426 mutant ( 56 , 57 ) were obtained from Dr. A.M. Pendergast (Duke University Medical Center, Durham, NC). WT STAT5B and STAT5B dominant-active mutant (DAM) (H295R+S715F) were cloned into the pMX-puro retroviral vector carrying the puromycin-resistance gene ( 58 ). COOH-terminal truncated STAT5B (ΔSTAT5) dominant-negative mutant (DNM) was a gift from Dr. A. Mui (DNAX, Palo Alto, CA) ( 48 ). The mutant was cloned into the pMX-puro vector. Constructs were electroporated into 32Dcl3 growth factor–dependent murine myeloid precursor cells ( 59 ) growing in IMDM-CM (IMDM supplemented with 10% FBS, 2 mM l -glutamine, penicillin/streptomycin [100 μg/ml each], and 15% WEHI-conditioned medium [WEHI-CM] as a source of IL-3). BCR/ABL and/or STAT5B-expressing clones were obtained after selection in G418 (1 mg/ml)- or puromycin (2 μg/ml)-containing medium, respectively, and were maintained in IMDM-CM. Expression of BCR/ABL in G418-resistant mixed cell transfectants and in individual clones was confirmed by Western blot analysis with an anti-ABL antibody (see below). Ectopic expression of STAT5B-WT and STAT5B-DAM in the clones was confirmed by reverse transcription (RT)-PCR using primers spanning the EcoRI cloning site in the construct, since STAT5 expression was similar in the clones and in the nontransfected cells. In addition, STAT5B-DAM expression in transfected cells was confirmed by electrophoretic mobility shift assay (EMSA) detecting STAT DNA binding activity in growth factor– and serum-starved cells. Infections were performed as previously described ( 21 , 60 ) with some modifications. In brief, helper-free retroviruses were generated by transiently transfecting retroviral vectors into BOSC23 cells as described ( 61 ). 24 h after transfection, 5 × 10 6 bone marrow cells from C57BL/6TacfBR mice (The Jackson Laboratory ) treated 6 d before cell harvest with 5-fluorouracil (150 mg/kg body wt) were added to the monolayer of BOSC23 cells transfected with BCR/ABL- and/or STAT5B-containing vectors or with the insert-less vector and cocultivated for 72 h in 4 ml of IMDM supplemented with 10% FBS, l -glutamine, penicillin/ streptomycin in the presence of recombinant IL-3, Kit ligand, and IL-6 (Genetics Institute), and polybrene (2 μg/ml) as previously described ( 21 , 60 ). Freshly established BCR/ABL-positive 32Dcl3 clones were infected in the presence of IL-3 by cocultivation with BOSC23 cells transfected with STAT5B-DNM containing vector or with insert-less vector. Bulk cultures of hematopoietic cells obtained 72 h after infection were used for the experiments. Expression of BCR/ABL was confirmed by Western blot analysis with anti-ABL antibody. STAT5B-WT expression was documented by RT-PCR using primers spanning the EcoRI cloning site. Expression of STAT5B-DAM was monitored by EMSA assay and by RT-PCR using primers spanning the EcoRI cloning site. Expression of STAT5B-DNM (ΔSTAT) was detected by RT-PCR using primers spanning the NotI cloning site in the construct thus allowing amplification of a fragment corresponding to the ectopically expressed C-terminal truncated STAT5B, and by Western blot analysis with anti-STAT5 antibody raised against aa 451–649 . Moreover, STAT5B-DNM was shown to inhibit BCR/ABL-induced STAT5 DNA binding activity in the transfected cells, and the samples in which the inhibition was >75% were used in the studies. Expression of BCR/ABL and STAT5 was detected in total cell lysates by Western blot analysis with anti-ABL (Oncogene Science) and anti-STAT5 (Oncogene Science; Santa Cruz Biotechnology, Inc. ; and Transduction Labs.) antibodies, respectively. Tyrosine phosphorylated proteins and actin were detected using anti-phosphotyrosine (P.Tyr) antibodies (Upstate Biotechnology, Inc.; and Oncogene Science) and anti-actin ( Santa Cruz Biotechnology, Inc. ), respectively. Cells were starved of growth factors and serum for 3 h. BCR/ABL kinase activity was determined in anti-ABL immunoprecipitates from parental 32Dcl3 cells and wild-type or mutant BCR/ABL-expressing cells, using 5 μg enolase as substrate ( 62 ). Ras activation was determined by measuring GTP-bound Ras as previously described ( 15 ). The DNA binding activity of STAT was examined by EMSA as previously described ( 9 ) using the FcγRI GAS motif as a probe. The sequence of the probe and the nonspecific competitor were as described ( 9 ). Supershift assays were performed using anti-STAT1, -STAT3, and -STAT5 mAbs ( Santa Cruz Biotechnology, Inc. ). STAT5-dependent transactivation was examined by luciferase assay ( 36 , 48 ). In brief, Tk − ts13 hamster fibroblasts were cotransfected with the expression vector (wild-type or mutant) BCR/ABL or with the insert-less vector along with the STAT5-responsive luciferase reporter construct (β-casein-Luc) and the expression plasmids for STAT5B and β-galactosidase (β-Gal). 36 h after transfection, cells were starved from serum (0.1% BSA) for 48 h and harvested for the luciferase assay using Dual-Luciferase Reporter Assay System ( Promega ) according to the manufacturer's protocol. For each transfection, luciferase activity was normalized using β-Gal activity as an internal control. Susceptibility to apoptosis induced by growth factor and/or serum withdrawal was measured as described ( 17 ) with some modifications. In brief, cells (2 × 10 5 /ml) were incubated in IMDM supplemented with 2 mM l -glutamine, penicillin/streptomycin (100 μg/ml each), and 0.1% BSA or 10% FBS for 24 and 48 h. The percentage of apoptotic cells was determined by using the TACS1 Klenow in situ apoptosis detection kit (Trevigen) according to the manufacturer's protocol. Cells (10 6 ) were starved of IL-3 (10% FBS) or IL-3 and serum (0.1% BSA) for 12 h and cell cycle analysis was performed as previously described ( 63 ) using propidium iodide to stain DNA. This parameter was determined using a 5-bromo-2′-deoxyuridine (BrdU) detection and labeling kit ( Boehringer Mannheim ). In brief, cells were starved of IL-3 (10% FBS) or IL-3 and serum (0.1% BSA) for 8 h and then incubated with 10 μM BrdU for the next 4 h. Cytospin slides were prepared and fixed in 94.5% ethanol containing 5% acetic acid and 0.5% Triton X-100. BrdU was detected by staining with mouse anti-BrdU mAb followed by sheep anti–mouse IgG-FITC. Nuclei were counterstained with 500 μg/ml bisbenzimide H33258 ( Sigma Chemical Co. ). Slides were mounted in Vectashield mounting medium (Vector Labs.) and the percentage of BrdU-positive cells was determined using a Zeiss microscope equipped for epifluorescence mode. 500 cells per cytospin were counted from randomly selected fields. ICR SCID male mice (Taconic Farms, Inc.) were injected intravenously with 5 × 10 6 32Dcl3 cells expressing the indicated BCR/ABL and/or STAT proteins. 4 wk later, four to six mice per group were killed and organs were analyzed macroscopically and microscopically for the presence of leukemia ( 21 ). Terminally ill mice were also killed and examined for the development of leukemia. Tissue sections from bone marrow, spleen, liver, lungs, kidneys, and brain were fixed in phosphate-buffered formalin and embedded in paraffin blocks. Two levels from each block were cut and stained with hematoxylin and eosin. In addition, selected slides were stained for chloroacetate esterase (Leder stain) to assess myeloid differentiation of blast cells. To investigate mechanisms and functional consequences of STAT activation by BCR/ABL, we first identified BCR/ABL mutants defective in STAT activation. Based on the findings that v-Src requires an intact kinase domain and both SH3 and SH2 domains to stimulate STATs ( 43 ), the following BCR/ABL mutants were used: the kinase-deficient K1172R mutant; the ΔSH2 mutant lacking the entire SH2 domain ; the SH2 FLVRES motif mutant R1053L; the ΔSH3 mutant lacking the entire SH3 domain ; the SH3 domain mutant P1013L; the ΔSH3+ΔSH2 mutant lacking both the SH3 and SH2 domains ; the P1013L+R1053L mutant carrying single aa substitutions in both the SH3 and the SH2 domains; and the ΔSH3+R1053L and P1013L+ΔSH2 mutants lacking either the SH3 or the SH2 domain and containing a single aa substitution in the other domain. After transfection of growth factor–dependent 32Dcl3 murine myeloid precursor cells, G418-resistant clones were selected and examined for the expression of BCR/ABL proteins . G418-resistant BCR/ABL-expressing cell clones (three to six different clones for each BCR/ABL mutant) were then starved of growth factors and serum and nuclear extracts were examined for activation of STAT DNA binding activity using the STAT binding site from the FcγRI promoter as a probe. Cells expressing full-length BCR/ABL (WT) but not the control parental 32Dcl3 cells, or cells expressing the K1172R kinase-deficient BCR/ ABL mutant showed STAT DNA binding activity . The specificity of the DNA-protein complex was investigated by EMSA in which an excess of unlabeled specific or nonspecific oligonucleotide was used as competitor. The DNA–protein complex was not detectable, or was markedly inhibited in the presence of 100- or 10-fold excess of specific competitor, respectively (data not shown). STAT1, STAT3, STAT4, STAT5, and STAT6 have previously been shown to bind efficiently to the probe, and STAT1, STAT3, and STAT5 have been reported to be activated by p210 BCR/ABL ( 24 – 27 , 55 ). In supershift assays performed to determine which STATs were involved in DNA-protein interaction, most of the complex was supershifted by the anti-STAT5 antibody, whereas no supershift was detected in the presence of the anti-STAT3 or the anti-STAT1 antibody (data not shown). In addition, when anti-STAT immunoprecipitates were blotted with the anti-P.Tyr antibody, STAT5 phosphorylation was prominent, STAT1 was weakly phosphorylated, and STAT3 was not detected in its phosphorylated form (data not shown). Thus, STAT5 represents the majority of the STAT activity induced by BCR/ABL, in accordance with other reports ( 24 – 27 ). BCR/ABL mutants deleted in either the SH3 (ΔSH3) or the SH2 (ΔSH2) domain, or those carrying a single aa substitution in the SH3 or the SH2 domain , were able to activate STAT5 DNA binding activity . However, deletion of both the SH3 and the SH2 domains (ΔSH3+ ΔSH2 BCR/ABL mutant) completely abolished the ability to activate STAT5 . Lack of STAT5 activation was also observed in cells expressing the P1013L+ΔSH2 BCR/ABL mutant, whereas ΔSH3+R1053L and P1013L+ R1053L BCR/ABL mutants activated STAT5 . Phosphorylation of STAT5 in IL-3 and serum-starved 32Dcl3 cells expressing wild-type and mutant BCR/ABL proteins was monitored by Western blot analysis of anti-STAT5 immunoprecipitates with anti-P.Tyr antibody and correlated with STAT5 DNA binding activity . Lack of STAT5 activation in clones expressing ΔSH3+ ΔSH2 or P1013L+ΔSH2 BCR/ABL mutants was not due to a general metabolic defect of the transfected cells, because it was fully restored by addition of IL-3 (data not shown). Parental 32Dcl3 cells and cells transfected with kinase-deficient K1172R BCR/ABL mutant expressed full-length and COOH-terminal truncated STAT5 proteins, as indicated by detecting two distinct bands in Western blotting (using the anti-STAT5 antibody raised against aa 451–649) of anti-STAT5 immunoprecipitates obtained with an antibody raised against the NH 2 terminus (aa 5–24) of STAT5 . The upper band was also detected by an antibody raised against the COOH terminus (aa 763–779) of STAT5B (data not shown). Interestingly, the COOH-terminal truncated STAT5 was usually detected in parental 32Dcl3 cells and in cells expressing the kinase-deficient BCR/ABL mutant but not in cells expressing wild-type BCR/ABL or the various SH3+SH2 BCR/ABL mutants retaining kinase activity . To examine the ability of BCR/ABL mutants to modulate the transactivation activity of STAT5, a reporter plasmid carrying the luciferase gene under the control of the STAT5-regulated β-casein promoter ( 36 , 48 ) was coexpressed in Tk − ts13 cells with wild-type STAT5 and the various BCR/ABL plasmids, and the transactivation ability of STAT5 was measured by luciferase assay. Stimulation of STAT5 transactivation activity by BCR/ABL (wild-type and mutants) correlated with their ability to induce STAT5 phosphorylation and DNA binding . Lack of STAT5 activation in cells expressing ΔSH3+ ΔSH2 or P1013L+ΔSH2 BCR/ABL mutants was not due to the inability of these mutants to function as tyrosine kinase, since similar (albeit not identical) patterns of tyrosine phosphorylated proteins were detected in lysates of cells expressing wild-type BCR/ABL or SH3+SH2 BCR/ABL mutants unable to activate STAT5 , and immunoprecipitated BCR/ABL mutants were able to phosphorylate enolase in in vitro kinase assays . Likewise, lack of STAT5 activation was not the consequence of detectable defects in Ras activation since the SH3+SH2 BCR/ABL mutants, like WT BCR/ABL, were all able to activate Ras . STAT5 activation–deficient BCR/ABL mutants failed to protect 32Dcl3 cells from apoptosis induced by withdrawal of IL-3 (10% FBS) or of IL-3 and serum (0.1% BSA) . The BCR/ABL SH3+SH2 mutants , which retain the ability to activate STAT5, did not protect from apoptosis in IL-3– and serum-deprived medium (0.1% BSA), as only a minority of cells (15–25%) were alive after 48 h of culture . However, these mutants provided significant protection from apoptosis (50–60% of viable cells) in the presence of serum and the surviving cells readily adapted to growth factor–free culture conditions and proliferated without IL-3 (data not shown). To determine if inhibition of STAT5 activity interferes with the antiapoptotic effects of BCR/ABL, freshly established 32Dcl3 clones expressing BCR/ABL were infected with a retrovirus carrying STAT5B-DNM and were assessed 72 h later for apoptosis in cultures deprived of IL-3 (10% FBS) or IL-3 and serum (0.1% BSA). Approximately 60% of the cells infected with the STAT5B-DNM retrovirus underwent apoptosis 48 h after growth factor and/or serum withdrawal . Cell cycle analysis after 12 h of starvation from IL-3 (10% FBS) or IL-3 and serum (0.1% BSA) revealed a reduced proportion of cells in S phase and an increase in the fraction of G0/G1 phase cells in STAT5 activation–deficient BCR/ABL mutant expressing cells compared with those expressing BCR/ABL WT or BCR/ABL SH3+SH2 mutants capable of activating STAT5 . Moreover, BrdU incorporation assay showed a reduced rate of DNA synthesis in cells expressing STAT5 activation–deficient BCR/ABL SH3+SH2 mutants , as compared with those expressing BCR/ABL WT or BCR/ABL SH3+SH2 mutants activating STAT5 . The changes in cell cycle distribution and DNA synthesis were dependent on the expression of mutant BCR/ABL and not on differences in growth abilities of the clones, because all clones showed similar proliferation rate in the presence of IL-3 (data not shown). Inhibition of STAT5 function in BCR/ABL-positive 32Dcl3 cells by transient expression of STAT5B-DNM caused a reduction of the percentage of cells in S and G2/M phase and an increase of the percentage of cells in G0/G1 phase in comparison to cells expressing BCR/ABL or BCR/ABL and WT STAT5B (data not shown). Lack of STAT5 activation by BCR/ABL mutants correlated with their reduced leukemogenic potential. SCID mice injected with 32Dcl3 cells expressing WT BCR/ABL died after 4–7 wk due to leukemia as confirmed at necropsy. Mice inoculated with cells expressing BCR/ABL SH3 and/or SH2 mutants activating STATs succumbed to leukemia after 10–20 wk . By contrast, mice injected with cells expressing STAT5 activation– deficient BCR/ABL mutants did not develop leukemia after 20 wk, as confirmed by histopathological examination of four mice per group, and remained leukemia-free during a 6-mo observation period. Inhibition of STAT5 activity by transient transfection of the dominant-negative mutant impaired the leukemogenic potential of BCR/ABL-expressing 32Dcl3 cells . SCID mice injected with BCR/ABL-positive STAT5B-DNM transient transfectants survived longer (10–17 wk) than those inoculated with cells coexpressing BCR/ABL and WT STAT5B (5–7 wk). To determine whether STAT5 is essential for the antiapoptotic activity of BCR/ABL, STAT5B-DAM or STAT5B-WT was introduced into 32Dcl3 parental cells or cells expressing BCR/ABL SH3+SH2 mutants defective in STAT5 activation. 32Dcl3 cells expressing STAT5B-DAM, but not cells expressing STAT5B-WT, were transiently resistant to apoptosis induced by IL-3 and/or serum withdrawal , but all died after 72–96 h. Cell cycle analysis after 12 h of starvation revealed that 32Dcl3 cells expressing STAT5B-DAM did not proliferate in the absence of IL-3 and/or serum . In contrast, STAT5B-DAM permanently rescued the apoptotic phenotype of IL-3 and/or serum-starved 32Dcl3 cells expressing STAT5 activation– deficient BCR/ABL SH3+SH2 mutants , stimulated DNA synthesis and cell cycle progression in serum- and/or IL-3–free medium , and allowed IL-3–independent proliferation (data not shown). To determine whether restoration of growth factor independence by STAT5B-DAM in 32Dcl3 cells expressing the ΔSH3+ΔSH2 or P1013L+ΔSH2 BCR/ABL mutant would also rescue their leukemogenic potential, SCID mice were injected intravenously with double-transfected cells and monitored for the development of leukemia. 4 wk after injection, organs taken from the mice were evaluated by visual inspection and light microscopy for the presence of leukemia. Consistent with previous studies ( 9 ), injection with WT BCR/ABL-expressing cells resulted in extensive leukemia involving both hematopoietic and nonhematopoietic organs in all four mice examined. Mice injected with 32Dcl3 cells expressing the ΔSH3+ΔSH2 or the P1013L+ΔSH2 BCR/ABL mutant showed no evidence of leukemia at 4 wk after cell inoculation (six and four mice per group examined, respectively) and remained alive after a 6-mo observation period. Similarly, mice injected with cells expressing STAT5B-DAM also showed no signs of leukemia at 4 wk after inoculation (five mice tested) and remained alive after 6 mo of observation. In contrast, all nine mice injected with 32Dcl3 cells coexpressing STAT5B-DAM and the ΔSH3+ΔSH2 or P1013+ΔSH2 BCR/ABL mutant developed leukemia within 4 wk. In bone marrow, spleen, liver, lungs, and kidneys, the leukemia resembled that caused by WT BCR/ABL, but was less extensive, e.g., liver typically showed scant periportal and sinusoidal infiltrates, and lungs showed patchy interstitial infiltrates without formation of frank, large tumors. ΔSH3+ΔSH2 or P1013L+ΔSH2 BCR/ABL- and STAT5B-DAM coexpressing cells more frequently involved meninges (six out of nine) and even brain parenchyma (two out of nine), as compared with BCR/ABL WT-expressing cells, which only occasionally and focally involved meninges (one out of four mice) at 4 wk after leukemia cell injection. Leukemic cells in the terminally ill mice injected with cells coexpressing BCR/ABL mutant and STAT5B-DAM formed invasive parenchymal foci within the central nervous system (CNS) of 13 out of 15 animals analyzed, whereas only small meningeal foci were observed in 12 out of 20 terminally ill mice injected with BCR/ABL WT-expressing cells. Mice inoculated with cells expressing ΔSH3+ΔSH2 or P1013L+ΔSH2 BCR/ABL mutants and STAT5B-DAM died of leukemia after 7–11 wk, whereas cells transformed with BCR/ABL WT killed the mice in 4–7 wk . To determine whether STAT5 plays an essential role in BCR/ABL-mediated transformation of bone marrow cells, we tested whether STAT5B-DNM inhibits transformation of hematopoietic progenitor cells by WT BCR/ ABL, and whether STAT5B-DAM rescues the impaired transformation potential of ΔSH3+ΔSH2 and P1013L+ ΔSH2 BCR/ABL mutants. Bone marrow cells from 5-fluorouracil–treated mice were infected with retroviral vectors carrying the various BCR/ABL and/or STAT5B cDNAs, and transformation was assessed by growth factor–independent colony formation ( 21 , 60 ). In the presence of threshold concentrations of recombinant murine IL-3 (0.1 U/ml), a high number of colonies formed from marrow cells infected with a retrovirus encoding WT BCR/ABL; fewer colonies formed in the absence of IL-3 . Coinfection of marrow cells with a STAT5B-WT or STAT5B-DAM induced a moderate increase in BCR/ABL-dependent colony formation , whereas coinfection with STAT5B-DNM inhibited colony formation induced by WT BCR/ABL by ∼60% and ∼75% in the presence or absence of IL-3, respectively. Upon infection of mouse bone marrow cells with retroviruses carrying STAT5 activation–deficient BCR/ABL mutants , no hematopoietic colonies formed in methylcellulose in the absence of IL-3 and only few (as in control groups) developed in cultures supplemented with IL-3 . However, coinfection of marrow cells with the retroviruses carrying the ΔSH3+ΔSH2 or P1013L+ΔSH2 BCR/ABL mutant and STAT5B-DAM, but not STAT5B-WT, induced large (data not shown), and numerous colonies. Infection of marrow cells with the retrovirus carrying the STAT5B-DAM alone did not stimulate the formation of growth factor– independent colonies . In addition, expression of STAT5B-DAM did not rescue the transformation-deficient phenotype of the K1172R kinase-deficient BCR/ABL mutant or of the BCR/ABL TM Y177F+ R522L+Y793F , both of which failed to activate STAT5 in 32Dcl3 cells and in bone marrow cells . Activation of STAT5 is detected in Philadelphia 1 (Ph 1 ) cell lines ( 23 – 26 ), in hematopoietic cell lines ectopically expressing BCR/ABL ( 27 ), and in CML primary cells ( 55 ). However, the mechanism(s) of BCR/ABL-dependent activation of STAT5 and its role in BCR/ABL leukemogenesis are essentially unknown. In this study, we investigated the mechanism(s) of BCR/ABL-regulated STAT5 activation and assessed its role in BCR/ABL leukemogenesis. In 32Dcl3 cells transfected with various BCR/ABL mutants, STAT5 activation was dependent on intact SH3 and SH2 domains that might be required for a direct interaction with STAT5 or with intermediate molecules linking structurally and/or functionally BCR/ABL and STAT5. Since only a small amount (if any) of BCR/ABL was usually detectable in complex with STAT5 (data not shown), it is possible that the BCR/ABL-STAT5 complex is too unstable for ready detection or that the interaction of BCR/ABL with intermediate molecules is the primary mechanism of STAT5 activation. In their COOH-terminal portions, STAT5A and STAT5B have two proline-rich regions that may be recognized by the SH3 domain of BCR/ABL or by an adaptor protein(s) serving as a bridge between BCR/ABL and STAT5. The interaction between the STAT5 P.Tyr(s) and the BCR/ABL SH2 domain does not seem to be involved in STAT5 activation, because the R1053L substitution in the FLVRES motif of the BCR/ABL SH2 domain, which reduces the ability of the SH2 domain to bind P.Tyr ( 64 ), did not interfere with STAT5 activation even when the BCR/ABL SH3 domain was deleted or carried the P1013L mutation, which impairs the interaction with proline-rich motifs ( 65 ). However, deletion of the entire SH2 domain from BCR/ABL in the context of a mutant lacking the SH3 domain or carrying the P1013L substitution prevented BCR/ABL-dependent STAT5 activation, suggesting that a portion of the BCR/ABL SH2 domain, distinct from the P.Tyr binding motif (FLVRES), is essential for this effect. One possibility is that the STAT5 SH2 domain recognizes one of the tyrosines in the BCR/ABL SH2 domain, or that the BCR/ABL SH2 domain interacts with STAT5 or with another intermediary protein(s) in a non-P.Tyr-dependent manner ( 57 ). The BCR/ABL SH3+SH2 domains may also create a “pocket” required for the direct or indirect activation of STAT5. There is evidence to suggest intramolecular contact between the BCR/ABL SH3 and SH2 domains, collaboration between these domains, and mutual functional influence of one domain on the other ( 66 ). The signaling pathways stimulated by BCR/ABL SH3 and SH2 domains that lead to STAT activation are unknown. RIN1, the Ras binding protein ( 67 ) that interacts simultaneously with both the SH3 and SH2 domains of BCR/ABL ( 68 ), might not be involved in this process because it does not coimmunoprecipitate with STAT5 (data not shown). The PI-3k/Akt pathway, which is affected by mutation/deletion of the BCR/ABL SH2 domain ( 21 ), is also probably not essential for STAT activation because no STAT activation was detected in 32Dcl3 cells expressing Akt dominant-active mutants (data not shown). A recent report showed that the COOH-terminal portion of the v-ABL oncogene is required for the interaction with JAK1 and the activation of STAT5 ( 69 ), suggesting that BCR/ABL and v-ABL use different mechanisms to activate STAT5. Unlike v-Abl, BCR/ABL neither associates with nor phosphorylates JAK1 or JAK2 in hematopoietic cells (24, 26, 55, and our unpublished results). The mechanism(s) of BCR/ABL-dependent activation of STAT5 seems similar to that induced by v-SRC. Like BCR/ABL, v-SRC does not phosphorylate JAKs in 32Dcl3 cells ( 43 ). In addition, BCR/ABL-STAT5 and v-SRC-STAT5 complexes are not readily detectable (26, 43, and this paper), and both oncoproteins require the SH3 and the SH2 domains to activate STATs (43 and this paper). Perhaps the characterization of proteins interacting with the SH3-SH2 segment of BCR/ABL may shed light on the mechanism(s) of STAT5 activation, even if other BCR/ABL domains might be important in this process. The role of STAT5B in BCR/ABL-dependent protection from apoptosis and induction of growth factor–independent proliferation was assessed after perturbation of STAT5 activity using two different strategies: (a) coexpression of WT BCR/ ABL and STAT5B-DNM; or (b) coexpression of STAT5 activation–deficient BCR/ABL mutants and STAT5B-DAM. Transient expression of STAT5B-DNM induced apoptosis and arrested cell cycle progression in the majority of BCR/ABL-expressing cells cultured in the absence of serum and/or IL-3. This partial inhibitory effect could be due to expression levels of STAT5B-DNM insufficient to block STAT5 activity in some cells. However, it is more likely that more than one independent pathway is required for the reduced apoptosis susceptibility and the growth factor-independent proliferation of BCR/ABL-expressing cells. Indeed, the dominant-active STAT5B mutant induced transient protection from apoptosis in 32Dcl3 cells, but upon expression in cells cotransfected with STAT5 activation–deficient BCR/ABL SH3+SH2 mutants , the double-transfectants were permanently protected from apoptosis and proliferated in a growth factor–independent manner. Several proteins such as Bcl-2 ( 22 ), Bcl-X L (70), Ras ( 71 ), and mitochondrial Raf ( 72 ) have been implicated in the transduction of antiapoptotic signals generated by BCR/ABL. Bcl-2 downmodulation was observed in cells expressing various BCR/ABL SH3+SH2 domain mutants, regardless of their ability to activate STAT5 (data not shown). The expression of functional GTP-bound Ras was upregulated in cells expressing STAT5 activation-deficient BCR/ABL mutants (this paper). Also, STAT5 was activated in cells expressing the Δ176–426 BCR/ABL mutant, which neither activates mitochondrial Raf-1 nor protects transfected cells from apoptosis induced by IL-3 withdrawal ( 72 ). There might be a functional link between STAT5 and Bcl-X L , as the Bcl-X L promoter contains a STAT5 binding site ( 73 ); however, the expression of Bcl-X L was not regulated by BCR/ABL in growth factor–starved 32Dcl3 cells (data not shown). Thus, BCR/ABL-dependent activation of STAT5 may involve a novel antiapoptotic mechanism(s). Our data on the antiapoptotic effect of STAT5-DAM are consistent with previous reports indicating that STAT5 plays an important role in antiapoptotic pathways regulated by IL-2 ( 34 ) and that STAT5 regulates the expression of the A1 protein ( 52 ), an antiapoptosis member of the Bcl-2 family ( 74 ). The ΔSH3+R1053L or P1013L+R1053L BCR/ABL mutant was able to activate STAT5 but protected only 50– 60% of the cells from apoptosis induced by IL-3 withdrawal. Reduced susceptibility to apoptosis of 32Dcl3 cells expressing STAT5 activation-deficient BCR/ABL SH3+SH2 mutants and STAT5B-DAM, in comparison to cells expressing BCR/ABL SH3+ SH2 mutants activating endogenous STAT5 , may be due to several reasons. First, ectopically expressed STAT5B-DAM may stimulate its downstream effectors more effectively than endogenous STAT5 activated by BCR/ABL mutants. In accordance with this possibility, A1 mRNA levels appear to be higher in clones expressing the BCR/ABL ΔSH3+ ΔSH2 mutant and STAT5B-DAM than in clones transfected with the BCR/ABL ΔSH3+R1053L mutant (data not shown). Second, proapoptotic signaling molecules such as SHIP ( 75 ) could be differentially regulated by the various BCR/ABL SH3+SH2 mutants ( 76 ) and might interfere with antiapoptotic activity of STAT5. Third, proteins modulating STAT5 activity such as CRKL ( 77 ) or CIS ( 78 ) could be differentially activated in hematopoietic cells by the BCR/ABL SH3+SH2 mutants. Cells expressing STAT5-activating BCR/ABL SH3+ SH2 mutants and surviving in growth factor–free medium became readily growth factor independent. Thus, although not sufficient, STAT5 activation might be necessary for BCR/ABL- dependent cell cycle progression ( 79 ). Indeed, expression of STAT5B-DAM in cells transfected with STAT5 activation-deficient BCR/ABL SH3+SH2 mutants not only restored protection from growth factor deprivation–induced apoptosis, but also significantly increased the percentage of cells synthesizing DNA, suggesting that STAT5 synergizes with STAT5 activation–deficient BCR/ABL SH3+SH2 mutants to stimulate growth factor–independent proliferation. However, it cannot be excluded that survival signals induced by constitutively active STAT5 are necessary to complement the mitogenic signals stimulated by the STAT5 activation–deficient BCR/ABL SH3+SH2 mutants. Some of the 32Dcl3 clones expressing STAT5 activation–defective BCR/ABL SH3+SH2 mutants became competent for STAT5 activation after long-term culture. This phenomenon was associated with the emergence of growth factor independence, and could reflect the use of an alternative signaling pathway(s) by BCR/ABL mutant ( 80 , 81 ), the reactivation of an autocrine loop for growth factor production which has been shown to require an intact BCR/ABL SH2 domain ( 82 , 83 ), or the occurrence of secondary mutations ( 84 ) that may induce constitutive activation of STAT5. The requirement for STAT5 activation in BCR/ ABL-induced abrogation of IL-3 dependence in 32Dcl3 cells is consistent with similar data in v-SRC–transfected cells ( 43 ). The importance of STAT5 in cell cycle progression was previously suggested, based on the suppression of DNA synthesis and proliferation in an IL-3–dependent BaF3 cell line expressing a STAT5 dominant-negative mutant ( 48 ). Moreover, STAT5B-DAM induced IL-3–independent proliferation of BaF3 cells ( 58 ). Also, bone marrow progenitors from STAT5A and STAT5A+STAT5B knockout mice demonstrate a defect in granulocyte-macrophage colony-stimulating factor–induced proliferation ( 32 , 52 ). The involvement of STAT5 activation in BCR/ABL leukemogenesis was demonstrated by showing that: (a) expression of a dominant-negative STAT5B mutant suppressed the leukemogenic potential and the transformation (growth factor–independent colony formation) of BCR/ABL-expressing 32Dcl3 cells or bone marrow cells, respectively; and (b) a dominant-active STAT5B mutant rescued leukemogenic potential and growth factor–independent colony formation of 32Dcl3 cells or primary bone marrow cells, respectively, expressing STAT5 activation-defective BCR/ABL SH3+SH2 mutants. BCR/ABL-dependent leukemogenesis of 32Dcl3 cells and transformation of primary mouse marrow cells was markedly, but not completely, inhibited by coexpression of dominant-negative STAT5B mutant. The simplest explanation for this partial inhibitory effect is that expression levels of STAT5B-DNM were insufficient to completely block STAT5 activity. Indeed, BCR/ABL-induced STAT5 DNA binding activity was not completely inhibited by coexpression of the dominant-negative STAT5 mutant. However, in light of the results of the complementation assays (see below) it seems likely that the full potential of BCR/ ABL to transform bone marrow cells rests in the activation of several independent pathways. The rescue of the leukemogenic potential of the STAT5 activation–deficient SH3+SH2 BCR/ABL mutant by dominant active STAT5 was not complete, as indicated by the slower development of disease in mice injected with mutant-expressing cells than in mice injected with cells expressing WT BCR/ABL. Most likely, other signaling pathways, such as the PI-3k/Akt pathway, are affected by deletion/mutation in the SH3+SH2 domains of BCR/ABL ( 21 ), and are not rescued by STAT activity. The higher frequency of CNS involvement, compared with other organs, in mice injected with mutant BCR/ ABL and STAT5B-DAM coexpressing cells might be due to STAT5-dependent transactivation of a gene(s) required for homing to CNS. On the other hand, the earlier death from leukemia in mice injected with WT BCR/ABL- expressing cells might explain the apparent lower frequency of CNS involvement since large leukemic infiltrates in the CNS would not have formed at the time of necropsy. The role of STAT5 in BCR/ABL-mediated transformation was also confirmed in transformation (growth factor–independent colony formation) assays with primary mouse bone marrow cells. STAT5 activation–deficient BCR/ABL SH3+SH2 mutants were completely defective in transformation; expression of dominant-active STAT5B mutant rescued the transformation potential of these BCR/ABL mutants, but not of the kinase-deficient K1172R BCR/ABL. However, the impaired leukemogenic potential of SH3+SH2 domain BCR/ ABL mutants is not only due to lack of STAT5 activation. Indeed, the ability of STAT5-activating BCR/ABL SH3+SH2 mutants to transform murine bone marrow cells was significantly diminished (data not shown), in accordance with previous findings indicating that intact SH3 and SH2 domains are required for BCR/ABL-mediated transformation ( 9 , 21 ), and that STAT5B-DAM restored most but not all transformation potential of STAT5 activation–deficient BCR/ABL SH3+SH2 mutants . Therefore, other signaling pathways regulated by BCR/ABL SH3+SH2 domains, in addition to STAT5 activation, are required for the full transformation potential of BCR/ABL. Interestingly, the transformation potential of the Y177F+R522L+Y793L BCR/ABL TM, which also does not activate STAT5, was not rescued by coexpression of STAT5B-DAM. This probably reflects the inability of activated STAT5 to rescue a BCR/ ABL mutant defective in certain signaling pathways. For example, the STAT5 activation–deficient BCR/ABL SH3+ SH2 mutants were competent for Ras activation, whereas the BCR/ABL TM was unable to induce an increase of functional GTP-bound Ras ( 56 ), suggesting the requirement for both Ras and STAT5 activation in the leukemogenic potential of BCR/ABL. The mechanism(s) whereby STAT5-regulated pathways are involved in BCR/ABL-dependent transformation of hematopoietic cells is unknown, but the involvement of STAT5 effectors with a potential role in proliferation, survival, and transformation can be postulated based on the identification of several STAT5-regulated genes. For example, STAT5 induces expression of the A1 gene, a member of the Bcl-2 family that protects 32Dcl3 cells from apoptosis ( 74 , 85 ). Bfl-1, the human homologue of A1, cooperates with the E1A oncogene in transformation ( 86 ), raising the possibility that A1 is activated by STAT5B-DAM in cells expressing STAT5 activation–deficient BCR/ABL SH3+ SH2 mutants, and functions as a mediator of the leukemogenic potential of these double-transfectants. The proto-oncogene pim-1 is also regulated by STAT5 ( 48 ) and is involved in leukemic transformation ( 87 ). Also, Bfl-1 (A1) and pim-1 reportedly collaborate with c-myc ( 87 ) and c-myb ( 88 ), which are required for BCR/ABL transformation ( 89 , 90 ). In conclusion, this study demonstrates that intact BCR/ ABL SH3 and SH2 domains are required for the induction of STAT5 activity and that a STAT5-dependent pathway(s) plays a crucial role in BCR/ABL leukemogenesis. | Study | biomedical | en | 0.999997 |
10209041 | Various cell lines were maintained in standard culture media supplemented with l -glutamine, nonessential amino acids, sodium pyruvate, penicillin/streptomycin, and fetal bovine serum. A rabbit anti–human (h)GrpL serum was generated in this study against a His-Tag fusion protein containing the SH2 domain of hGrpL as described below. Antiphosphotyrosine 4G10 and anti-LAT/pp36/38 were purchased from Upstate Biotechnology, Inc. Anti–SLP-76, anti-Grb2, anti–SH2 domain–containing protein tyrosine phosphatase 2 (SHP-2), anti-Sos1, and anti-Sos2 were purchased from Santa Cruz Biotechnology . 64.1 (anti-CD3) was provided by Dr. John Hansen (Fred Hutchinson Cancer Research Center, Seattle, WA), C305 (anti-TCR) was provided by Dr. G. Koretzky (University of Iowa, Iowa City, IA), and OKT3 (anti-CD3) was obtained from the American Type Culture Collection. A full-length cDNA encoding hGrpL was identified through random sequencing of clones from a library prepared from the acute myelogenous leukemia cell line KG-1a. Overlapping partial cDNA clones encoding murine (m)GrpL were isolated by low stringency reverse transcriptase (RT)-PCR using multiple hGrpL-specific primer sets and murine splenic total RNA as the template. RT-PCR at low annealing temperature was performed according to the Titan™ One Tube RT-PCR System protocol ( Boehringer Mannheim Inc. ). PCR products were resolved on 1.5% agarose gels. cDNA bands corresponding in sizes to the estimated lengths of hGrpL were excised from the gel and eluded using the QIAEX II Gel Extraction Kit (QIAGEN Inc.) according to the suppliers protocol. Isolated cDNAs were then ligated into a T/A cloning vector (Invitrogen) and transformed into competent E . coli (Invitrogen). Recombinant clones containing mGrpL inserts were selected by low stringency hybridization to a hGrpL cDNA probe and then sequenced. This allowed for the identification of primer sets specific for mGrpL for the isolation of cDNA clones encoding both the 5′ and 3′ ends of mGrpL by rapid amplification of cDNA ends. The gene specific primers chosen were: 5′ cDNA end, 5′-CTG AGC TTT CGC ACT GGA GAC ATT CTG; 3′ cDNA end, 5′-GCA TCA GCG TGG CGT TCA CCT CAC TTC C. The Marathon-Ready™ cDNA kit ( Clontech Laboratory Inc. ) was used for RACE PCR and performed as recommended by the supplier. The resulting PCR products were cloned into T/A cloning vectors (Invitrogen) and sequenced. To generate the GrpL SH2 His-Tag fusion protein, PCR was used to amplify a piece of cDNA encoding the SH2 domains of GrpL using the full-length GrpL cDNA as template and the following primer pairs: forward primer, 5′-GCG G GG ATC C CC CAG TTT CCC AAA TGG TTT CAC; reverse primer, 5′-GCG GG G AAT TC C TCT GTC TCT AAG GAA GAT CTG. Underlined nucleotides represent BamHI and EcoRI sites in the forward and reverse primers, respectively. PCR products were cut with these enzymes and ligated into BamHI/EcoRI cut pET-28c(+) (Novagen, Inc.). To generate the GST fusion construct, the insert from the His-Tag fusion construct was cut out by BamHI/EcoRI and subcloned into BamHI/EcoRI cut pGEX-5X-2 ( Amersham Pharmacia Biotech ). The fusion protein constructs were then transformed into E . coli strain BL21(DE3) and induced with IPTG for protein production. To generate the Myc-His tagged hGrpL expression plasmid the coding region of hGrpL was amplified using the following primer pairs: forward primer, 5′-CCT GAA G CT CGA G TC GGG TCA TGG GTG CCA CGT A; reverse primer, 5′-GCG GAA TTC GGA GGA TGG AAG CTG TTG CCA AGT TT. Underlined nucleotides represent XhoI and EcoRI sites in the forward and reverse primers, respectively. The PCR product was cloned into the pcDNA3.1(+)/Myc-His A vector (Invitrogen), in-frame with the c-Myc epitope and poly-His tags. The hGrpLMyc-His cassette was then excised with BamHI and PmeI and subcloned into the pEF expression vector (provided by Dr. G. Koretzky) at the BamHI and filled-in XbaI sites (pEF-GrpL). The pEF–SLP-76 and the NF-AT luciferase reporter (NF-AT Luc) constructs were also gifts from Dr. G. Koretzky. To detect GrpL message in cell lines, ∼30 μg of RNA were resolved on 1.2% morpholine propanesulfonic acid (MOPS)/formaldehyde agarose gels, transferred onto GeneScreen Plus membranes (NEN™ Life Science Products, Boston, MA), and fixed by UV cross-linking. Multiple tissue Northern blots were obtained from Clontech . Northern blots were prehybridized and hybridized to either the full-length GrpL cDNA or β-actin probes labeled with α-[ 32 P]dCTP (NEN™ Life Science Products) according to the manufacturer's instructions. RT-PCR was performed using the Titan™ One Tube RT-PCR System ( Boehringer Mannheim ). The reaction was carried out using 1 μg of total RNA and 20 nM forward and reverse primers. Primers used were: forward primer, 5′-TGG AAG CTG TTG CCA AGT TTG ATT TCA C; reverse primer, 5′-CTT CTC GGG TTC TGT CTC TAA GGA AG. This primer set amplifies a 452-bp fragment containing the NH 2 -terminal SH3 and the SH2 domains from cells expressing GrpL message. The amount of total RNA used was monitored by amplification of a 1-kb fragment encoding G3DPH using the following primer set: forward primer, 5′-TGA AGG TCG GAG TCA ACG GAT TTG GT; reverse primer, 5′-CAT GTG GGC CAT GAG GTC CAC CAC. Cycling parameters PCR were programmed according to the instructions provided with the Titan™ system ( Boehringer Mannheim ). Jurkat T cells were pelleted and resuspended at 5–10 × 10 6 /ml in medium. Cells were allowed to equilibrate at 37°C for 15 min and then stimulated with either 5 or 10 μg/ml of anti-CD3 or 2.5 mM of H 2 O 2 plus 250 μM of sodium orthovanadate (referred to as pervanadate). Either class-matched antibodies or medium only was used as control stimulus. Stimulation was terminated by dilution of cell suspensions into >10 vol of ice-cold PBS. Cells were pelleted and lysed in a buffer containing 150 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 0.5% NP-40, protease inhibitors (2 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin), and phosphatase inhibitors (10 mM NaF, 1 mM Na 3 VO 4 , and 5 mM Na 4 P 2 O 7 ). Lysates were clarified by centrifugation before immunoprecipitation. Immunoprecipitation, SDS-PAGE, and Western blot analysis were conducted as described ( 35 ). Binding of primary antibodies to blots was detected with horseradish peroxidase–conjugated secondary antibodies or streptavidin (Jackson ImmunoResearch Labs.) and an enhanced chemiluminescence kit ( Amersham Pharmacia Biotech ). Jurkat cells in log-phase were washed with serum-free RPMI 1640 once and electroporated in triplicate with a total of 60 μg of plasmid at 240 V, 960 μF using a Gene Pulser (Bio-Rad) at 10 7 cells/400 μl serum-free RPMI 1640. In general, 20 μg of NF-AT Luc were cotransfected with either 20 μg of pEF, pEF–SLP-76, pEF-GrpL, or pEF–SLP-76 plus pEF-GrpL. Sonicated salmon sperm DNA ( Sigma Chemical Co. ) was used to maintain the total DNA at 60 μg whenever necessary. After electroporation, cells were incubated in fully supplemented RPMI 1640 for 14–18 h at 37°C, 5% CO 2 . Cells were then transferred into 96-well plates at 2–5 × 10 5 cells/ well in a total of 100 μl medium. Cells were stimulated in triplicate with 1:500 dilution of C305 anti-CD3 for 6–8 h at 37°C. Control cells were maintained in culture medium. After stimulation, cells were lysed in 50–100 μl Reporter Lysis buffer ( Promega ) for 15 min at room temperature. Luciferase activity was quantified by adding 10 μl of the lysate to 50 μl of luciferase assay substrate ( Promega ) and immediately measured with a GenProbe Leader™ 1 luminometer (Wallac Inc.). To normalize for variations in transfection efficiency, luciferase activities in different transfection conditions were expressed as a percentage of maximal NF-AT responses determined by treating cells with 50 ng/ml PMA and 1 μM ionomycin as previously described ( 18 , 20 ). Through random sequencing of cDNA clones in a library prepared from the acute myelogenous leukemia cell line KG-1a, we identified a cDNA encoding a 330-amino acid polypeptide with a predicted molecular weight of ∼37 kD. The linear amino acid sequence of this polypeptide organizes into an NH 2 -terminal SH3 domain followed by an SH2 domain, a proline-rich region, and finally a COOH-terminal SH3 domain . A comparison to other known proteins available in current sequence data banks revealed that this protein is most similar to the adaptor proteins, Grb2 and Grap . Based on this similarity, we have designated this molecule, GrpL for Grb2-related protein of the lymphoid system (see below). Using RT-PCR and a combination of 5′- and 3′-RACE (Materials and Methods), we also isolated the full-length cDNA encoding the murine homologue of GrpL. Human and murine GrpL are highly conserved proteins sharing >90% identity in the SH3 and SH2 domains (Table I ). The least conserved area is the proline-rich region with ∼74% identity (Table I ). Amino acid sequence alignment between hGrpL, Grb2, and Grap revealed that hGrpL is 38–56% identical to Grb2 and Grap in the SH3 and SH2 domains . This conservation is slightly less than that between Grb2 and Grap (49–68%; reference 33 ). The biggest difference between GrpL and Grb2 or Grap is the presence of a proline-rich region between the SH2 and COOH-terminal SH3 domain. This proline-rich region could potentially be bound by SH3 domain–containing proteins found in other adaptor molecules ( 36 , 37 ). Taken together, the amino acid composition of GrpL suggests that it may interact with different proteins in lymphocytes when compared with Grb2 or Grap. The transcripts for Grb2 are expressed ubiquitously in mammalian species ( 38 ), whereas Grap mRNA is expressed at considerably higher levels in lymphoid tissues than in other tissues ( 33 , 34 ). Northern blot analysis of mRNA isolated from different human tissues demonstrated that the transcripts for GrpL are relatively restricted to hematopoietic tissues. Thus, transcripts of 1.4 kb and 3–3.5 kb were detected in lymph node, bone marrow, spleen, thymus, and peripheral blood lymphocytes, as well as weakly in testis, but not in stomach, thyroid, spinal cord, trachea, adrenal gland, prostate, ovary, or small intestine . Probing total RNA from a panel of cell lines by Northern blot analysis and RT-PCR further confirmed the restricted expression pattern of GrpL . GrpL transcripts were detected in the myeloid and erythroid progenitor cell lines KG-1a and K562, respectively, but not in the myeloid cell line HL60. In T cells, GrpL is expressed in the Jurkat, Molt-4, HSB-2, CEM, and HPB-ALL lines, but not in the HuT78 line. Interestingly, of all the B cell lines examined, GrpL transcripts were only found in EBV-transformed lymphoblastoid cell lines including T5-1, HCLL7678, CESS, F4, FBM2-4, and IM9. Other B cell lines, including the Burkitt's lymphomas (Daudi, BJAB, and Ramos), the pre-B cell lines (REH, NALM-6, and BLIN1), the sIgM + early B cell lines (BLIN1 and 1E8), and the myeloma cell line RPMI8226, did not express any GrpL transcripts. Results from the above experiments show that GrpL expression is highly regulated and restricted to subsets within hematopoietic lineages. To characterize the GrpL protein and the proteins with which it interacts, we generated a rabbit hetero-antiserum specific for GrpL by immunizing with a His-Tag GrpLSH2 fusion protein. This antiserum can specifically immunoprecipitate from Jurkat T cells two proteins ∼38 and 40 kD in size . Both proteins were detectable by Western blot analysis of either GrpL immunoprecipitates (IPs) or whole cell lysates. This antiserum does not cross-react with Grb2, which runs at a lower molecular weight on gels , and by all criteria it appears to be specific for GrpL. To detect proteins potentially capable of interacting with GrpL, we pharmacologically stimulated Jurkat cells with pervanadate, immunoprecipitated GrpL, and then Western blotted with either antiphosphotyrosine or anti-GrpL sera . 2–5 min after stimulation, a major tyrosine phosphorylated protein 40 kD in size and a minor 38-kD phosphoprotein were detectable in anti-GrpL but not control serum IPs. This doublet most likely corresponds to GrpL itself, which migrates to the same position on gels ; it is possible that these bands represent differentially phosphorylated forms of GrpL. Compared with pervanadate, TCR ligation induced only weak tyrosine phosphorylation of GrpL (data not shown). In addition to the 38–40-kD doublet, a second major tyrosine phosphorylated protein ∼68–70 kD in size was also detected in the GrpL IP after pervanadate treatment . In T cells, substrates for protein tyrosine kinases in this size range include SLP-76, SHP-2, Syk, and ZAP-70. Indeed, in the GrpL IP obtained from both unstimulated and pervanadate-stimulated Jurkat cells, we were able to detected SLP-76 but not ZAP-70 (data not shown). However, the amount of SLP-76 associating with GrpL remained relatively unchanged after stimulation. Moreover, the SLP-76 molecule coimmunoprecipitated with GrpL before stimulation showed an almost undetectable level of tyrosine phosphorylation . These results are consistent with the idea that GrpL can interact with SLP-76 independently of tyrosine phosphorylation as has been suggested for Grb2 and SLP-76 ( 21 , 27 ). The increased signal of the 68–70-kD molecule coimmunoprecipitated with GrpL after stimulation may be a consequence of increased tyrosine phosphorylation of SLP-76 and/or the recruitment of additional tyrosine phosphorylated proteins of the same size to GrpL. In the SLP-76 IP obtained from pervanadate-stimulated Jurkat cells, we could also detect tyrosine-phosphorylated protein at 38– 40, 50–55, and 110 kD . To examine the possibility of distinct functional roles played by different Grb2-like molecules in T cells, we asked whether GrpL and Grb2 interact with different proteins. We immunoprecipitated either GrpL or Grb2 from Jurkat cells and probed for associating proteins. SLP-76 was detected in GrpL IPs , as also shown in Fig. 3 C. However, we could not detect any SLP-76 associating with Grb2. Consistent with this was the presence of GrpL but not Grb2 in the SLP-76 IPs . On the other hand, we were able to detect Sos1 and Sos2, GEFs of Ras, in Grb2 IPs , as has been reported in other cell systems. Interestingly, no detectable Sos1 or Sos2 was found to be coimmunoprecipitated with GrpL . These results suggest that in Jurkat T cells, GrpL preferentially complexes with SLP-76, whereas Grb2 preferentially complexes with Sos1 or Sos2. Since both Grb2 ( 10 – 14 ) and Grap ( 33 , 34 ) can associate with tyrosine-phosphorylated LAT/pp36/38 upon TCR ligation, we asked whether GrpL could also associate with tyrosine-phosphorylated LAT/pp36/38. Jurkat cells were stimulated by anti-CD3, and LAT/pp36/38 was immunoprecipitated from NP-40 Jurkat cell lysates at various time points after stimulation. As reported before ( 24 ), tyrosine-phosphorylation of LAT/pp36/38 increased dramatically after TCR ligation, and started to decline after 3 min of stimulation. This was correlated with the appearance of Grb2 in the LAT/pp36/38 IPs . However, although the levels of GrpL proteins in NP-40 Jurkat cell lysates remained unchanged throughout stimulation, we consistently did not detect any GrpL associating with LAT/pp36/38 in these lysates . Since SLP-76 has been shown to regulate NF-AT activation in T cells ( 18 , 20 , 39 ), we investigated whether GrpL could play a role in this pathway. NF-AT activation was monitored by the expression of a NF-AT luciferase reporter construct in Jurkat cells. As described before, overexpression of SLP-76 by cotransfection of a SLP-76 expression vector augmented the anti-CD3–induced NF-AT activation ( 18 , 20 , 39 ). Overexpression of GrpL alone consistently augmented anti-CD3– induced NF-AT activation . Moreover, an additive effect between GrpL and SLP-76 on NF-AT activation was observed when both were overexpressed in Jurkat cells. Since various SH2 domains can interact with a wide spectrum of tyrosine-phosphorylated targets, we tested to which tyrosine-phosphorylated protein(s) the SH2 domain of GrpL could bind. When the GST/GrpLSH2 fusion protein was used to probe pervanadate-stimulated Jurkat cells, tyrosine-phosphorylated proteins that migrated at 36, 69, and 71 kD could be readily detected . In some experiments, we also detected 95- and 130-kD tyrosine-phosphorylated proteins. The GST/GrpLSH2 fusion protein also precipitated a similar pattern from lysates prepared from anti-CD3–stimulated Jurkat cells . By analogy to Grb2 ( 24 ), these proteins could be LAT, ZAP-70, SHP-2, Vav, and perhaps c-Cbl. As shown in Fig. 7 B, we were able to confirm that SHP-2 was among the phosphoproteins precipitated by the GST/GrpLSH2 fusion protein from pervanadate-activated Jurkat cells. Although tyrosine-phosphorylated SLP-76 also migrates at ∼70 kD, we consistently did not detect any SLP-76 precipitated by GST/GrpSH2. Critical functions are played by a variety of adaptor molecules in linking receptor proximal signal transduction events such as activation of receptor-associated kinases to downstream effector molecules. In this report, we describe the identification and characterization of a new member of the Grb family of adaptor proteins. GrpL shares similar structural organization as Grb2 and Grap. A distinguishing feature of GrpL not found in Grb2 or Grap is the presence of a 100-amino acid stretch connecting its SH2 and SH3C domains. Although this region is the least conserved between mGrpL and hGrpL, it does contain multiple proline and glutamine residues at conserved positions . Thus, two PxxP motifs are found in both hGrpL and mGrpL. In hGrpL, they are P191TLP and P202QPP; in mGrpL, they are P190LGP and P210QPP. These polyproline motifs conform to the consensus sequence recognized by different SH3 domains ( 36 , 37 ). Accordingly, it is likely that the proline residues in GrpL do constitute a proline-rich domain capable of interacting with as yet to be defined SH3-containing proteins. In contrast to the broadly expression Grb2, GrpL expression is restricted to hematopoietic cells . In this regard, GrpL is similar to Grap that transcripts for both are found at much higher levels in hematopoietic tissues ( 33 , 34 ). Although Grap is preferentially expressed in blood cells, little is known about its expression in different stages of T and B cell maturation or in different lymphocyte subsets. GrpL transcripts are expressed at lower levels in the bone marrow than in the secondary lymphoid organs. Importantly, the highest levels of GrpL transcripts were detected in the thymus , suggesting that GrpL may play a crucial role in regulating early T cell development. Northern blot analysis on cell lines also demonstrates that GrpL transcripts are not expressed in all lymphocytes , suggesting that their expression may be modulated during lymphocyte ontogeny by specific signals. To test this possibility, we fractionated normal B cells into subsets and measured levels of GrpL transcripts. A clear difference was observed in GrpL transcript expression in naive but not in germinal center B cells (Solow, S.A., A.J. Marshall, T.J. Yun, M.K. Ewings, and E.A. Clark, manuscript in preparation). To elucidate the signals involved in switching the expression of GrpL on and off will provide further insights into the functions of GrpL and its associating proteins at different stages of lymphocyte development. We have identified one of the GrpL-interacting proteins to be SLP-76 . Interaction between GrpL and SLP-76 appears to be constitutive, since it could be detected in both stimulated and unstimulated T cells . Accordingly, this interaction may not require the tyrosine phosphorylation of either SLP-76 or GrpL. In support of this, results presented in Fig. 3 C show that the amount of SLP-76 coimmunoprecipitating with GrpL remained relatively constant, although SLP-76 became heavily tyrosine-phosphorylated after pervanadate treatment. Hence, our data argue against the possibility that the SH2 domain of GrpL mediates its interaction with SLP-76. Grb2 has been reported to be an adaptor molecule capable of associating with SLP-76 ( 24 ). In these studies, GST fusion proteins containing Grb2 or Grb2 SH3 domains recognized SLP-76 from lysates of resting or activated T cells independent of its tyrosine-phosphorylation ( 21 , 27 ). Using deletional mutants of SLP-76, the Grb2 binding site was subsequently mapped to the central proline-rich region of SLP-76 from amino acid residues 224–244 ( 21 ). These results implicate the SH3 domain of Grb2 to be the primary mediator for interaction. Based on the similar structure between GrpL and Grb2 and the phosphorylation-independent interaction between GrpL and SLP-76, it is likely that the SH3 domain(s) of GrpL also mediates its interaction with SLP-76. Experiments are currently in progress to examine this possibility. Several recent studies have provided strong evidence for an essential function served by SLP-76 in T lineage cells. Biochemical experiments show that SLP-76 can interact with both positive and negative regulators. TCR ligation induces ZAP-70 to phosphorylate SLP-76 at three NH 2 -terminal tyrosine residues ( 20 , 30 , 39 ). Once phosphorylated on its NH 2 -terminal tyrosine residues, in particular Y113 and Y128, SLP-76 can be bound by the SH2 domain of Vav ( 18 , 19 ). Overexpression of SLP-76 augment IL-2 promoter activity ( 21 ), and SLP-76 also acts synergistically with Vav to augment IL-2 promoter activity ( 18 ). Moreover, the biological activity of SLP-76 is absolutely dependent on an intact proline-rich region, and hence, an interaction with adaptor proteins containing SH3 domain(s) ( 21 ). On the other hand, the COOH-terminal SH2 domain of SLP-76 binds to a 130-kD tyrosine-phosphorylated protein ( 40 , 41 ). This protein has been cloned, designated to be SLAP-130/Fyb, and may be involved in downregulating the activating function of SLP-76 on the IL-2 promoter ( 41 ). The most compelling evidence to argue for an indispensable in vivo function of SLP-76 in T lineage cell is provided by the recent gene targeting experiments. SLP-76 − / − mice are characterized by an early arrest in T cell development. CD4 + /CD8 + , CD4 + , and CD8 + thymocyte populations are completely absent from thymuses of SLP-76 knockout mice, resulting in a corresponding lack of peripheral T cells ( 22 , 23 ). In the SLP-76 − / − mice, TCR-β chain genes appear to undergo normal rearrangement and exogenous anti-CD3 fails to drive the generation of double positive cells. Therefore, it is proposed that the absence of SLP-76 may render the pre-TCR complex fail to deliver maturational signals to developing T cells ( 22 , 23 ). Our observation that GrpL associates with SLP-76 in vivo suggests GrpL plays a pivotal role in TCR-mediated signal transduction cascades. Studies from different laboratories have convincingly demonstrated binding of recombinant Grb2 or Grb2 SH3 domains to SLP-76 both in cell lysates and on Western blots ( 9 , 21 , 27 ), but the presence of SLP-76 in Grb2 IPs and vice versa have not been reported thus far. Our data argue that SLP-76 is more likely to associate with GrpL than Grb2 under physiological conditions in T cells . This GrpL/SLP-76 association may regulate signaling pathway(s) involving SLP-76 in T cells. One downstream target for SLP-76 is NF-AT. SLP-76 and Vav can synergistically enhance anti-CD3–induced NF-AT activation in T cells ( 18 ). Results in Fig. 6 demonstrate that overexpression of GrpL alone is sufficient to enhance the TCR-induced NF-AT. More importantly, GrpL cooperated with SLP-76 in this pathway as suggested by the additive effect upon overexpression of both GrpL and SLP-76. Given the functions of SLP-76 in regulating T cell development and gene expression through its interaction with the GEF Vav, we propose that GrpL may also modulate the guanine nucleotide exchange activity of Vav via its association with SLP-76 to regulate T cell development and functions. By contrast, Grb2 may function principally via the Sos1 and Sos2 GEFs. Another feature that further distinguishes GrpL from Grb2 is the lack of association between GrpL and tyrosine-phosphorylated LAT/pp36/38 in NP-40 lysates prepared from TCR-activated T cells . Detergent-insoluble membrane subdomains known as glycolipid-enriched microdomains or detergent-insoluble rafts concentrated with lipid-linked signaling proteins have been proposed to be focal areas from which receptor-mediated signaling originates ( 42 – 44 ). Recently, components of the activation TCR complex, including tyrosine-phosphorylated LAT, have been localized to such detergent-insoluble rafts, and disruption of these rafts inhibits signaling via the TCR ( 45 , 46 ). Since GrpL appears to be an integral part of the TCR-mediated NF-AT activation pathway , it is possible that GrpL may also be localized to rafts upon TCR ligation. This may allow GrpL to interact with upstream molecules activated by the TCR, including tyrosine-phosphorylated LAT in rafts. Experiments are now in progress to examine this possibility. The in vitro binding of GrpLSH2 domain to tyrosine-phosphorylated SHP-2 suggests that SHP-2 may potentially interact with GrpL . However, we have not yet detected an in vivo association between GrpL and SHP-2 in activated T cells (data not shown). SHP-2 possesses the dual functions of either potentiating or inhibiting signal transduction depending on the receptor system involved. For example, SHP-2 has been shown to be required for coupling receptors to the activation of the MAPK pathway ( 47 , 48 ). In T cells, SHP-2 interacts with CTLA-4, which is believed to be the mechanism whereby CTLA-4 downregulates T cell activation, possibly via dephosphorylation of Shc ( 49 ). Nevertheless, a recent report suggests that SHP-2 may also be a positive regulator of TCR-mediated signaling. Thus, a multimeric complex involving Grb2, LAT/pp36/38, SHP-2, and a 110-kD tyrosine-phosphorylated protein has been reported in T cells ( 50 ). Interestingly, expression of a SHP-2 mutant devoid of phosphatase activity inhibits the TCR-induced MAPK pathway activation. Similar to Grb2, the SH2 domain of GrpL mediates binding to SHP-2. This binding is dependent on T cell activation and tyrosine phosphorylation of SHP-2 . Hence, after TCR ligation, the interaction between Grb2 and SHP-2 may facilitate the redistribution of the GrpL– SLP-76 complex to the plasma membrane. Consequently, SLP-76 would become available for phosphorylation by ZAP-70 to generate the Vav binding sites. Taken together, our data support a model in which distinct complexes of Grb2/Sos1/Sos2 and GrpL–SLP-76 may be responsible for connecting the TCR to the Ras- and Rho/Rac/CDC42-mediated effector pathways, respectively . Thus, Grb2 is the primary adaptor molecule responsible for the recruitment of the GEFs Sos1 and Sos2 for Ras ( 26 ) to LAT/pp36/38. The lymphoid-restricted, Grb2-like adaptor protein Grap can also serve a similar function by interacting with Sos and Ras ( 33 , 34 ). Recruitment of Ras to the inner plasma membrane activates distal MAPKs including Erk1 and Erk2 ( 26 ). On the other hand, Vav, a GEF for the Rho/Rac/CDC42 family of GTP-binding proteins expressed in lymphoid cells ( 51 , 52 ), binds to tyrosine-phosphorylated SLP-76 via its SH2 domain ( 9 ), and regulates NF-AT activation ( 18 ) and cytoskeletal reorganization in T cells ( 53 ). Since GrpL associates with SLP-76 and regulates NF-AT activation , one testable possibility is that a trimolecular interaction among SLP-76, Vav, and GrpL may be required for optimal NF-AT activation and reorganization of cytoskeleton during T cell activation. During the course of this study, two other groups independently identified the same gene and protein as we describe herein, which they have designated as Mona ( 54 ) and Gads ( 55 , 56 ). In vitro data suggest that in nonlymphoid cells, GrpL (Mona/Gads) may interact with either the M-CSF receptor, c-fms ( 54 ), or with c-kit ( 55 ). In agreement with our results, Liu et al. ( 56 ) independently found that GrpL can bind to SLP-76 and together with SLP-76 can promote the activation of NF-AT in T cells. We have recently identified another adaptor protein, BAM-32, which unlike GrpL , SLP-76 ( 18 – 21 ), or the B cell adaptor BLNK ( 57 ), inhibits the activation NF-AT (Marshall, A.J., H. Niiro, T.J. Yun, and E.A. Clark, manuscript in preparation). Thus, antigen receptor–induced activation of NF-AT in lymphocytes is carefully regulated by adaptor proteins. | Study | biomedical | en | 0.999997 |
10209042 | cGKI-deficient (cGKI −/− ) mice were generated as previously described ( 8 ) and bred and maintained at the animal facility of the Institut für Pharmakologie und Toxikologie (TU München). For experiments, 4–8-wk-old wild-type and cGKI −/− mice of either sex on 129sv background were used (litter- or age-matched animals). All experimental procedures performed on these mice were approved by the German legislation on protection of animals. Western blot analyses of cGKI and cAK catalytic subunits cAKα and cAKβ in platelets of wild-type, heterozygous, and cGKI −/− mice were carried out as previously reported ( 21 ). To assess the effect of cAK and cGK activation on VASP phosphorylation, platelet-rich plasma (PRP) was prepared as described below and incubated for 20 min at room temperature in the presence of 0.1% DMSO (control), or the specific activators of cAK and cGK, cBIMPS (0.1 mM; Biolog Life Science) and 8-pCPT-cGMP (0.1 mM; Biolog Life Science), respectively. Platelets were then pelleted and lysed in Laemmli buffer. VASP phosphorylation was assessed by the mobility shift of phosphoVASP ( 9 ) and detected by immunoblotting with a VASP-specific antibody (Dianova). 4–8-wk-old mice of either sex and genotype were anesthetized by chloroform inhalation and their chests were opened. After intracardial injection of buffer A (75 μl buffer [138 mM NaCl, 1.0 mM MgCl 2 , 2.9 mM KCl, 0.36 mM NaH 2 PO 4 , 20 mM Hepes, and 5 mM glucose, pH 7.4] containing heparin [300 U/ml]), blood was collected by cardiac puncture. Whole blood was diluted with buffer B (two parts buffer supplemented with 30 mg/ml BSA and 30 U/ml heparin), and centrifuged for 10 min at 100 g . PRP was carefully removed and replaced by an equal volume of buffer B. After mixing gently, samples were centrifuged again for 10 min at 100 g and PRP was removed. The resulting PRP fractions were pooled and centrifuged at 1,000 g for 10 min. The pellet was resuspended in buffer B to give a final density of 1–3 × 10 5 platelets/μl. To evaluate the role of cGKI in the regulation of platelet function in vitro (aggregation, shape change, and serotonin release), PRP was incubated for 20 min with 0.1% DMSO (control), 0.1 mM 8-pCPT-cGMP, or 0.1 mM cBIMPS. Aggregation was started by addition of collagen (5 μg/ml) and followed in an aggregometer (Chronolog). Aggregation and shape change were measured by recording the light transmission, as previously described ( 22 ). To assess platelet serotonin release, the PRP was loaded with 37 MBq/ml [ 3 H]5-hydroxytryptamine (5-HT; Amersham Healthcare) for 1 h at 37°C. Thereafter, the PRP was centrifuged for 10 min at 1,000 g , and the supernatant was removed. The pellet was resuspended in buffer A at a density of 1–3 × 10 5 platelets/μl. After 30 min at room temperature, platelets were incubated for 20 min at 37°C as indicated above and stimulated with collagen (5 μg/ml) for 5 min. After centrifugation, the amount of [ 3 H]5-HT released into the supernatant was determined by liquid scintillation counting. To study platelet adhesion/aggregation during I/R in vivo, blood (0.8–1.0 ml) from an anesthetized donor mouse of either genotype was collected by cardiac puncture and added to a solution containing 500 μl Ca 2+ - and Mg 2+ -free PBS (PAN Systems), 200 μl citrate buffer (100 mM dextrose, 2.6 mM citric acid monohydrate, and 2.7 mM tri-sodium citrate dihydrate) and 15 μl prostaglandin (PG)E 1 ( Sigma -Aldrich) (50 μg/ml). After addition of 100 μl fluorescent dye rhodamine 6G ( Sigma -Aldrich) (0.5 mg/ml) to label platelets ex vivo, the whole blood was centrifuged for 10 min at 100 g and PRP was carefully isolated. PRP was added to a solution containing 1,000 μl PBS (Ca 2+ and Mg 2+ free), 30 μl PGE 1 , and 200 μl citrate buffer. After centrifugation for 10 min at 1,000 g , the supernatant was removed and the resulting rhodamine-labeled pellet was resuspended in 500 μl PBS (Ca 2+ and Mg 2+ free). A 50-μl sample of the rhodamine-labeled platelet suspension was analyzed by using a flow cytometer (FACSort ® ; Becton Dickinson ) and an A C T counter (Coulter Corp.) to determine the purity and number of platelets, respectively. Labeled platelets (5.4 × 10 7 ) of either genotype were infused as bolus via the venous catheter. To evaluate the biological significance of cGKI in the regulation of platelet adhesion/aggregation in vivo, fluorescent platelets were infused after intestinal I/R and visualized in the postischemic microcirculation by intravital fluorescence microscopy. 4–6-wk-old inbred 129sv mice of either genotype (five to nine litter- or age-matched animals per group) were anesthetized by inhalation of isoflurane-N 2 O (0.35 FiO 2 , 0.015 liter/liter isoflurane; Forene ® ; Abbott GmbH). The animals were placed on a heating pad (Effenberger), and polyethylene catheters (Portex) were implanted into the left carotid artery and left jugular vein for continuous recording of mean arterial blood pressure and infusion of fluorescent platelets, respectively. After laparotomy, a segment of the jejunum was exteriorized and constantly superfused with 37°C Ringer's lactate. Segmental jejunal ischemia was induced for 60 min by occluding the supplying vessels with microsurgical clips. After reperfusion, the intestinal segment was exposed on a mechanical stage and platelet–platelet and platelet–endothelial cell interactions in the postischemic microvasculature were investigated by intravital microscopy. 15 min after the onset of the reperfusion, labeled platelets (5.4 × 10 7 ) of either genotype were infused as bolus via the venous catheter into the acceptor mouse of either genotype subjected to either I/R or sham operation. Platelet concentration in wild-type and cGKI −/− mice was 0.5 ± 0.02 × 10 6 ( n = 36) and 0.5 ± 0.06 × 10 6 ( n = 25)/μl blood, respectively. During the reperfusion, 10 nonoverlapping regions of interest from the submucosal vessels of the ischemic/reperfused segment were randomly selected in each mouse and observed for 30 s with a modified microscope (Leitz). The microscopic images with a final magnification on the video screen of 450× were recorded by a CCD camera connected to a video recording system (Sony Corp.). For analysis of platelet–platelet and platelet–endothelial cell interactions, a computer-assisted image analysis program (CAP IMAGE; Dr. Zeintl, University of Heidelberg, Heidelberg, Germany) and frame-to-frame analysis of the videotapes were used ( 23 ). All experiments were blinded, and adherence of platelets to the surface of arterioles and venules (vessel diameter, 15–85 μm) and formation of platelet aggregates in capillaries (diameter, 15 μm), arterioles, and venules (vessel diameter, 15–85 μm) were quantified. The number of adherent platelets was assessed by counting the platelets that did not move or detach from the endothelial surface within 15 s. Platelet adhesion is presented per square millimeter of endothelial surface. The number of occluding and nonoccluding aggregates was quantified within arterioles and venules and is presented per 100 vessels. To determine platelet aggregation in the capillary bed, the length (centimeter) of capillaries occluded by fluorescent platelets was measured and calculated per square centimeter of tissue cross-sectional area. To confirm the role of cGKI in the regulation of I/R-induced platelet adhesion/aggregation in vivo, the accumulation of 111 I-labeled wild-type or cGKI −/− platelets was assessed in the postischemic kidney. Platelets were pelleted from PRP (see above) and resuspended in 500 μl PBS (Ca 2+ and Mg 2+ free) containing 37 MBq/ml 111 I-oxine ( Amersham Healthcare). After incubation for 5 min at 37°C, the platelet suspension was centrifuged for 10 min at 1,000 g and the supernatant was removed. The resulting pellet was washed with 2 ml PBS (Ca 2+ and Mg 2+ free) and centrifuged for 10 min at 1,000 g . After removing the supernatant, the pellet was resuspended with 200 μl PBS (Ca 2+ and Mg 2+ free). Male wild-type 129sv mice were anesthetized using intraperitoneal injection of Avertin (1.2% tribromoethanol/amylalcohol in 0.9% saline solution) and placed on a heating pad for maintenance of body temperature. A polyethylene catheter (Portex) was implanted into the left jugular vein for infusion of 111 I-labeled platelets and for infusion of Ringer's lactate to maintain euvolemia. After a midline incision of the abdomen, the left renal artery and vein were isolated and subsequently occluded with a microsurgical clip for 30 min. 25 min after setting occlusion, 0.2 ml of washed, 111 I-labeled platelet suspension (2 × 10 7 ) of either genotype was infused via the venous catheter, and 5 min afterwards the clip was removed for reperfusion. After 25 min of reperfusion, the experiment was terminated and the kidney was removed, weighed, and homogenized. The homogenate was counted , and the accumulation of 111 I-labeled wild-type and cGKI −/− platelets after renal I/R was quantified as counts per minute per milligram wet weight of the kidney ( 24 ). To study the role of cGKI in the regulation of fibrinogen binding to agonist-stimulated wild-type and cGKI −/− platelets, blood (0.4–0.6 ml) was collected from wild-type ( n = 7) and cGKI −/− ( n = 5) mice by cardiac puncture. The platelets were separated as described above for intravital microscopy, with the exception that no rhodamine was added. The resultant pellet was resuspended in 6 ml of a solution containing equal parts PBS with and without Ca 2+ and Mg 2+ (PAN Systems). The purity and number of platelets were determined using the A C T counter. The wild-type or cGKI −/− platelets were preincubated at room temperature for 2 min with either PBS (PAN Systems), the NO-donor DEA-NO (100 nM final concentration; Alexis) or the stabile prostacyclin-analogue iloprost (10 μM final concentration, Ilomedin; Schering AG). After preincubation, the samples were stimulated with 0.2 U/ml mouse thrombin ( Sigma -Aldrich) or PBS, and immediately incubated for 10 min at room temperature with Alexa™ 488-conjugated fibrinogen (12.5 μg/ml final concentration; Molecular Probes). After incubation, all samples were fixed with 1% paraformaldehyde and the fluorescence intensity was analyzed using a flow cytometer (FACSort ® ; excitation at 488 nm, emission detection at 520 nm). The platelets were identified by their characteristic forward and sideward light scatter. Analysis of the fluorescence properties of 10,000 platelets was performed using a Lysis II data handling program ( Becton Dickinson ). The fluorescence intensity of unstimulated platelets, preincubated with DEA-NO, iloprost, or PBS (<15% of thrombin-stimulated fluorescence intensity), was subtracted from the fluorescence of the corresponding thrombin-stimulated sample. Data are presented as percentage of nonpretreated, thrombin-stimulated wild-type or cGKI −/− platelets. All data are presented as mean ± SEM. Statistical differences between two means were determined by Student's t test or Kruskal-Wallis test . P < 0.05 was regarded as significant, n indicates the number of animals. Deletion of the cGKI gene abolished immunoreactive cGKI protein without affecting the immunoreactive concentration of cAMP kinase . Platelet activation in response to collagen was similar in wild-type and cGKI −/− mice . In both wild-type and mutant platelets, collagen-induced shape change, aggregation, and serotonin release were prevented by activation of cAK by the cAK-specific cAMP-analogue cBIMPS . In contrast, activation of cGKI by the cGMP-analogue 8-pCPT-cGMP inhibited collagen-induced shape change, aggregation, and serotonin release in wild-type platelets, but had no effects on cGKI −/− platelets. This indicates that the effects of cGMP are mediated predominantly via activation of cGKI, whereas other cGMP receptors, such as phosphodiesterases and cyclic nucleotide-gated ion channels, play a minor role in the cGMP-dependent inhibition of platelet aggregation in vitro. Therefore, it is very likely that NO, which is known to elevate platelet cGMP levels ( 5 ), inhibits platelet adhesion and aggregation acting via cGKI. Cross-activation of cAK by cGMP does not appear to be operative in platelets. The reverse mechanism (i.e., activation of cGKI by cAMP, which has been postulated to mediate relaxation of smooth muscle cells; reference 25 ) is also not involved in the regulation of platelet function since the specific activator of cAK, cBIMPS, elicited a similar effect on both cGKI +/+ and cGKI −/− platelets. We therefore conclude that cGMP and cAMP signaling cascades inhibit platelet aggregation independent from each other. Several mechanisms might be involved in cGMP/cGKI-dependent inhibition of platelet aggregation. In various cell types, including platelets and smooth muscle cells, cGMP/ cGK lowers cytosolic Ca 2+ concentrations after stimulation, thereby affecting a variety of Ca 2+ -regulated processes (25– 28). Yet the details of cGMP/cGK-dependent regulation of platelet Ca 2+ homeostasis remain to be elucidated at the molecular level. Phosphorylation processes are likely to be involved in the antiaggregatory effects of cGMP/cGKI on platelets. To date, the only well-established substrate of cGK is VASP, a 46–50-kD vasodilator-stimulated protein, present in high concentrations in platelets ( 29 ). In the study reported here, VASP phosphorylation was assessed in wild-type and cGKI −/− platelets using the mobility shift of phosphoVASP ( 9 ). In murine platelets, VASP is phosphorylated in response to both cBIMPS and 8-pCPT-cGMP . The disruption of cGKI abolished cGMP-dependent in vivo phosphorylation of VASP in platelets without affecting VASP phosphorylation in response to the cAMP analogue cBIMPS. Although VASP phosphorylation correlates well with platelet inhibition, its precise functional role has not been established thus far. However, the subcellular location of VASP and the association with actin filaments and focal adhesions suggest a possible role in regulating platelet aggregation/adhesion ( 30 ). In fact, there is evidence to suggest that VASP phosphorylation is closely associated with the inhibition of the agonist-evoked activation of the fibrinogen-binding site of the glycoprotein IIb–IIIa ( 31 , 32 ), supporting a role of cGKI/VASP signaling in the regulation of platelet adhesion/aggregation. Growing evidence suggests that platelets play an important role in the pathogenesis of I/R-induced vascular injury and restenosis. Ischemia is associated with platelet accumulation early after the onset of reperfusion ( 17 ). Clinical and experimental studies have indicated that platelet adhesion and aggregate formation and the release of proinflammatory mediators from activated platelets in response to ischemia may impair restoration of nutritive blood supply during reperfusion ( 33 , 34 ). Although activation of cGKI attenuates platelet aggregation in vitro, the significance of the NO/cGMP/cGKI pathway in the homeostasis of platelet adhesion/aggregation during I/R in vivo has not been clearly defined thus far. The biological role of cGKI might be questioned, since the deficit in cGKI could be compensated by endothelium-derived prostacyclin signaling through the unperturbed cAK pathway ( 12 ). Therefore, to study the physiological relevance of cGKI in vivo, we determined platelet adherence and aggregation within the microcirculation of an ischemic/reperfused segment of the jejunum using intravital video microscopy. Wild-type and cGKI −/− mice (acceptor) were subjected to intestinal ischemia (60 min). After reperfusion, fluorescent wild-type and cGKI −/− platelets were infused and visualized within the intestinal submucosa (arterioles, capillaries, and venules) by intravital fluorescence microscopy. Since platelet adhesion to the injured vessel surface represents an early step in the process of platelet accumulation/ aggregation, we first determined the number of adherent platelets within arterioles and postcapillary venules of the postischemic submucosal microcirculation. Under control conditions without I/R (sham), wild-type platelets did not interact with wild-type endothelium . In contrast, numerous platelets were found firmly attached to the vascular wall of both arterioles and postcapillary venules in response to I/R . To assess the role of cGKI in the regulation of platelet function in vivo, cGK −/− platelets were transfused into cGK-deficient mice after I/R. The loss of cGKI drastically enhanced postischemic platelet–endothelial cell interactions. Within both arterioles and venules, the number of adherent platelets was increased four- to sixfold when compared with wild-type animals. Under control conditions, no adhesion of cGKI −/− platelets to the vessel wall was observed, indicating that a pathological stimulus, such as I/R, is required to induce platelet adhesion in cGKI −/− mice. Because platelets, endothelial cells, and smooth muscle cells express cGKI, we wanted to clarify whether cGKI expressed by platelets or present in the vascular wall is necessary to inhibit platelet adhesion during postischemic reperfusion. To determine the contribution of platelet cGKI, cGKI −/− platelets were infused into wild-type mice. The isolated loss of platelet cGKI dramatically enhanced I/R-induced platelet adhesion to the vascular wall of wild-type animals: 290 ± 45 and 181 ± 28 platelets were found firmly attached per square millimeter endothelial surface of arterioles and venules, respectively . Therefore, platelet adhesion is enhanced to a similar extent, independent of whether platelets alone or both platelets and vascular wall lack cGKI. Accordingly, I/R-induced adhesion of wild-type platelets was not increased in cGKI null mutants as compared with wild-type recipients . This demonstrates that the cGKI expressed in endothelium and smooth muscle cells plays a minor role in the regulation of platelet adhesion dynamics during I/R. During reperfusion, adherent platelets may subsequently aggregate leading to luminal narrowing and complete vascular (re-)occlusion, resulting in additional ischemia of the supplied tissue ( 18 – 20 ). To evaluate the participation of cGKI in the regulation of platelet aggregation in vivo, we quantified the presence of platelet aggregates in arterioles and venules of the postischemic jejunal segment. The number of occluding and nonoccluding aggregates in arterioles and venules was assessed within 10 randomly selected regions of interest and is presented per 100 vessels . Whereas aggregation of wild-type or cGKI −/− platelets was virtually absent under control conditions, platelets deficient in cGKI showed a very strong tendency to aggregate in response to I/R. In postischemic arterioles, 2–5 occluding and 17–26 nonoccluding aggregates were observed per 100 vessels when cGKI −/− platelets were transfused into wild-type or cGKI −/− mice, respectively . Likewise, the absence of cGKI in platelets enhanced aggregation in postcapillary venules (not shown). In contrast, no occluding or nonoccluding aggregates were detected when both platelets and the vascular wall expressed cGKI , indicating that cGMP/cGKI signaling pathways regulate both platelet adhesion and aggregation. To assess the role of cGKI present in endothelial cells and vascular smooth muscle, wild-type platelets were transfused into cGKI −/− mice . However, the absence of cGKI in the vascular wall did not significantly enhance the aggregation of wild-type platelets in arterioles or venules, suggesting that endothelial cell and smooth muscle cGKI are not involved in the regulation of platelet aggregation in vivo. Platelet aggregation in cGKI mutants was not confined to arterioles and venules, but was also frequently observed in capillaries (diameter, 10 μm). To assess the extent of platelet aggregation in the capillary bed of the postischemic submucosa, the length (centimeters) of capillaries occluded by fluorescent platelets was measured and calculated per square centimeter tissue cross-sectional area . Platelet aggregates in capillaries were nearly absent after transfusion of wild-type platelets into wild-type mice or cGKI mutants. In contrast, aggregation of cGKI −/− platelets in postischemic capillaries was a prominent phenomenon, independent of whether or not the vascular wall expressed cGKI. Hence, platelet adhesion and platelet aggregation in arterioles, capillaries, and venules are drastically enhanced when the platelets lack cGKI. In contrast, the absence of the endothelial/smooth muscle cGKI has no significant effects on homotypic platelet–platelet or heterotypic platelet–endothelium interactions in response to I/R. This indicates that under pathophysiological conditions, such as I/R, NO acts via the cGKI present in platelets to regulate platelet adhesion and aggregation in vivo. Although endothelial cells are considered to be the major source of NO, in particular under conditions associated with platelet activation, platelet-derived NO also plays an important role in regulating platelet aggregation and platelet recruitment ( 35 ). During aggregation, the NO release by platelets is significantly increased ( 35 ). This increase in NO formation can act via guanylyl cyclase to activate platelet cGKI. Therefore, platelets appear to have the ability to self-regulate their adhesion and aggregation upon activation by an autocrine/paracrine mechanism in which activated platelets release NO that acts on platelet cGKI to attenuate both adhesion and aggregation. It appears noteworthy that, under physiological conditions without I/R, platelet aggregation in arterioles, capillaries, and venules was absent in both wild-type animals and cGKI −/− mutants (not shown). This indicates that both endothelial cells and platelets are in an antiadhesive/anticoagulant state under physiological conditions and acquire a proadhesive/procoagulant phenotype in response to I/R. To evaluate whether the observed increase in postischemic platelet adhesion and aggregation due to the loss of platelet cGKI is confined to the intestine or rather a more general defect in platelet function, we analyzed the accumulation of wild-type and cGKI −/− platelets after I/R of the kidney. Wild-type animals were laparotomized, and the left renal artery and vein were cross-clamped for 30 min using microsurgical clips. 5 min before reperfusion, washed 111 I-labeled platelets were infused via a catheter implanted into the left jugular vein. After 25 min of reperfusion, the left kidney was excised and platelet accumulation was quantified as counts per minute per milligram wet weight. Renal I/R induced the accumulation of 111 Indium-labeled wild-type platelets in the postischemic wild-type kidney. However, platelets lacking cGKI exhibited a 46% higher accumulation in the wild-type kidney after I/R when compared with wild-type platelets , suggesting that cGKI inhibits postischemic platelet accumulation independent of the organ studied. The molecular mechanisms underlying cGKI-dependent inhibition of platelet adhesion/aggregation during I/R in vivo are as yet unclear. There is growing evidence indicating that I/R is associated with an activation of the platelet fibrinogen receptor, the glycoprotein (GP) IIb– IIIa ( 36 ). Binding of fibrinogen to the activated form of the platelet GP IIb–IIIa integrin complex plays a critical role in the process of platelet adhesion/aggregation ( 37 , 38 ). Although both NO and cGMP have been shown to interfere with agonist-evoked activation of the GP IIb–IIIa ( 31 , 39 ), the exact role of cGKI in the inhibition of GP IIb–IIIa activation has not been identified thus far. To determine whether cGKI mediates NO/cGMP-dependent regulation of GP IIb–IIIa function, we have investigated the effects of NO on agonist-induced fibrinogen binding to wild-type and cGKI −/− platelets in vitro. In wild-type platelets, pretreatment with NO decreased thrombin-induced fibrinogen binding by ∼46% . In contrast, in platelets lacking cGKI, the response to NO was nearly absent ( P < 0.05). Hence, cGKI-dependent inhibition of the GP IIb–IIIa adhesion complex is involved in the regulation of platelet adhesion/aggregation by NO/cGMP in vivo. The loss of platelet cGKI does not affect the ability of platelets to respond to prostacyclin. Preincubation with iloprost reduced agonist-induced fibrinogen binding to both wild-type and cGKI −/− platelets by ∼40%. Therefore, the inability of the endogenous cAMP kinase-activating system to compensate for the loss of cGKI in vivo is not due to alterations in the response of cGKI −/− platelets to exogenous prostacyclin. In conclusion, we have demonstrated that platelet cGKI attenuates agonist-induced platelet activation, fibrinogen binding, and aggregation in vitro. Moreover, the loss of cGKI in platelets is associated (a) with an increase in platelet accumulation in the postischemic kidney and (b) with a significant enhancement of both platelet adhesion and aggregation in the postischemic intestinal microvasculature in vivo. The platelet cAMP/cAK signaling cascade does not compensate for the loss of cGKI under pathophysiological conditions. Platelets are known to release NO upon activation; therefore, an autocrine/paracrine signaling cascade, including platelet-derived NO and platelet cGKI, is likely to be involved in the regulation of platelet adhesion and aggregation under physiological and, in particular, pathophysiological conditions. Since the accumulation and aggregation of platelets after endothelial injury is a common pathophysiological mechanism underlying many of the most important diseases, including myocardial infarction, angina pectoris, thrombotic stroke, and peripheral vascular insufficiency, specific activators of platelet cGKI might present a powerful strategy aimed at the prevention of I/R injury. | Study | biomedical | en | 0.999997 |
10209043 | Plasmids containing wild-type genes encoding HLA-C and -G proteins were used as described ( 27 , 28 ). Plasmids encoding chimeric HLA-Cw6 or HLA-Cw7 with the region encoding the conserved residue Arg 201 (near the beginning of the α3 domain) to the end of the mature protein replaced by that of HLA-G were also prepared as described ( 29 ). Proteins encoded by these plasmids were termed HLA-Cw6/ G-tail and HLA-Cw7/G-tail in the investigation of their relative turnover at the cell surface ( 29 ), but in the current report, they are renamed HLA-Cw6/G end and HLA-Cw7/G end to emphasize that these proteins contain part of the α3 domain and the complete transmembrane sequence as well as the cytoplasmic tail of HLA-G . Plasmid inserts encoding HLA-Cw6 truncated by the insertion of a stop codon were obtained by PCR from the plasmid encoding HLA-Cw6 in pcDNA3 and cloned back into pcDNA3 (Invitrogen Corp.) as KpnI–EcoRI fragments. The primers used for the PCR were 5′-GGGGTACCCCGCCGCCACCATGCGGGTCATGGCGCCCCGAACC-3′ for the 5′ end of HLA-Cw6, including an added KpnI restriction site and the Kozak sequence to enhance expression, coupled with each of the following for the 3′ end encoding the EcoRI restriction site and stop codon (underlined): 5′-CCGGAATTC TCA TCCACCTGAGCTCTTCCT-3′ for HLA-Cw6/315 stop, 5′-CCGGAATTC TCA CGCAGCCTGAGAGCAGCT-3′ for HLA-Cw6/325 stop, 5′-CCGGAATTC TCA GCCCTGGGCACTGTTGCT-3′ for HLA-Cw6/332 stop, and 5′-CCGGAATTC TCA CTCATCAGAGCCCTGGGC-3′ for HLA-Cw6/335 stop. To obtain the plasmid encoding HLA-Cw6 with cysteine at position 309 replaced by tryptophan, the residue in that position in HLA-A and HLA-G, two overlapping fragments were obtained by PCR using the plasmid encoding HLA-Cw6 in pcDNA3 as the template. The 5′ fragment was obtained using a T7 oligo ( New England Biolabs, Inc. ) as the 5′ primer together with 5′-GCTCTTCCTCCTCCACATCACAAC-3′, and the 3′ fragment was obtained using an SP6 oligo ( New England Biolabs, Inc. ) as the 3′ primer together with 5′-GTTGTGATGTGGAGGAGGAAGACC-3′. These two fragments were joined by PCR using SP6 and T7 oligos as primers and cloned into pcDNA3 as a KpnI–EcoRI fragment. The plasmid encoding HLA-Cw6 in which the transmembrane sequence had been replaced by that of HLA-G was made by using three fragments obtained by PCR from the plasmids encoding HLA-Cw6 and HLA-G. The 5′ fragment encoding the cytoplasmic tail of HLA-Cw6 was obtained by PCR using SP6 as the 5′ primer together with 5′-GCTGCTGTGCTGTGGAGGAGGAAGAGCTCA-3′. The fragment encoding the transmembrane domain of HLA-G was obtained by PCR using 3′-TGAGCTCTTCCTCCTCCACAGCACAGCAGC-5′ at the 3′ end and 5′-CTGAGATGGGAGCCATCTTCCCTGCCCACC-5′ at the 5′ end. These two fragments were then joined together by PCR using the appropriate flanking primers, producing a fragment encoding the cytoplasmic tail of HLA-Cw6 attached to the transmembrane domain of HLA-G. The 3′ fragment encoding the extracellular portion of HLA-Cw6 was obtained by PCR with T7 as the 3′ primer together with 5′-GGTGGGCAGGGAAGATGGCTCCCATCTCAG-3′. This fragment was then joined to the fragment encoding the cytoplasmic tail of HLA-Cw6 and transmembrane domain of HLA-G by PCR using T7 and SP6 oligos as primers. This insert was then cloned as a KpnI– EcoRI fragment into pcDNA3. All primers were purchased from Life Technologies and all plasmid inserts were sequenced by the Core Facilities, Dana-Farber Cancer Institute (Boston, MA) using T7, SP6, and primers internal to the class I MHC–encoded region. The human B cell line 721.221, deficient in cell surface expression of class I MHC proteins ( 30 ), was obtained from the American Type Culture Collection (ATCC). 721.221 cells were transfected with 100 μg of linearized plasmids by electroporation as described ( 28 ) and continually kept in the selection medium. Transfectants were sorted by flow cytometry to express equivalent levels of class I MHC protein detected by the conformation-specific mAb W6/32 ( 31 ). An exception, however, was that the cell surface expression of class I MHC protein in the HLA-G transfectant used was significantly higher than that of the other transfectants, as shown to be necessary for inhibition of NK cell cytotoxicity ( 27 , 32 ). Transfectants expressing the extracellular portions of HLA-C also expressed equivalent levels of the epitope for mAb L31 by flow cytometry. This mAb recognizes naturally occurring HLA-C heavy chains not associated with β 2 m ( 33 ). Transfectants were routinely stained with mAb W6/32 or G46-2.6 and analyzed by flow cytometry to ensure that equivalent cell surface expression of class I MHC proteins remained after time in culture. Transfectants also expressed equivalent levels of proteins previously found to be associated with class I MHC proteins (see Discussion), namely, CD20, CD45, CD53, CD81, CD82, HLA-DR, and IL-2R γ chain as determined by flow cytometry using appropriate antibodies. Expression of correctly sized heavy chains from HLA-Cw6, -Cw7, -G, -Cw6/G end, and -Cw7/G end associated with the light chain β 2 m was confirmed by immunoprecipitation from the transfectants with mAb BBM.1 that recognizes β 2 m ( 34 ) followed by SDS-PAGE analysis. cDNA made from mRNA extracted from these transfectants was used as a template for PCR of the class I MHC–encoding products, which were sequenced to further confirm the sequence of the appropriate MHC product in each transfectant. The cytolytic activity of NK lines and clones against various target cell lines and transfectants was assessed in 5-h 35 S-release assays as described ( 28 ). Assays were performed in duplicate or triplicate, and data values differed by <10% (and on average, ∼5%) of the mean. In all presented cytotoxicity assays, the spontaneous release of 35 S was <25% (and on average, ∼10%) of the maximal release. NK cell lines 986, I, 264, 268, and 532 were prepared from healthy donors as described ( 28 ). NK cell lines 15, 26, 53, 60, 69, 71, 86, and X1 were prepared from cells derived from healthy donors sorted to be CD56 + CD3 − by flow cytometry and plated at 30 cells/well (FACStar PLUS® ; Becton Dickinson ). These sorted cells were grown in X-Vivo 10 medium (Bio-Whittaker) supplemented with 0.1% PHA (Murex), 100 U/ml rIL-2 ( Boehringer Mannheim ), 10% lymphocult (Biotest), 5% human serum (Bio-Whittaker), 100 mM MEM sodium pyruvate (Life Technologies), 1% MEM nonessential amino acids (Life Technologies), 1% 2-ME (Life Technologies), and 50,000/well irradiated PBLs from two allogeneic donors and 10,000/well irradiated RPMI 8866. These sorted cells were restimulated with irradiated feeder cells once every 14 d. All NK lines and clones were periodically monitored to be CD3 − and CD56 + by antibody staining. NK cell cytotoxicity assays were performed at least 4 d after restimulation with feeder cells or fresh rIL-2 and were performed using only cells that appeared healthy through a standard laboratory microscope. mAb to NKIR1, EB6, and HP3E4 were purchased from Immunotech or were a gift from M. Lopez-Botet (Hospital Universitario de la Princesa, Madrid, Spain), respectively. mAb HP3E4 and EB6 both recognize p58 NKIR and can block NK cell recognition of HLA-C alleles belonging to group 1, including HLA-Cw6 and -Cw4, but can recognize serologically distinct epitopes ( 35 – 37 ). mAb recognizing other proteins were used as follows: HLA-C (L31; a gift from P. Giacomini and A.G. Siccardi, San Raffaele Scientific Institute, Milan, Italy), NKB1 (DX9; a gift from L. Lanier, DNAX), CD3 (SK7; Becton Dickinson ), CD20 (2H7; PharMingen ), CD45 (HI30; PharMingen ), CD53 (HI29; PharMingen ), CD56 (NCAM16.2; Becton Dickinson ), CD81 (JS-81; PharMingen ), CD82 (50F11; PharMingen ), CD94 (HP3D9; PharMingen ), HLA-A, B, C (G46-2.6; PharMingen and W6/32; ATCC), β 2 m (BBM.1; ATCC), HLA-DR (G46-6; PharMingen ), IL-2R γ chain . For mAb blocking of NKIR1 and CD94, TEPC183 ( Sigma Chemical Co. ) and GL183 (mAb to NKIR2; Coulter Immunology) were used as control IgM and IgG1 mAb, respectively. The Bsu36I restriction enzyme site in exon 4 of class I MHC genes was used to construct plasmids encoding chimeric proteins of HLA-Cw6 and -Cw7 attached to most of the α3, transmembrane, and cytoplasmic domains of HLA-G, termed HLA-Cw6/G end and HLA-Cw7/G end, respectively . These chimeric proteins were expressed in the HLA-A,B,C–negative B cell line 721.221 and compared with wild-type HLA-Cw6 and -Cw7 proteins for their capacity to inhibit lysis by various NK cell lines . NK lines 986 and I (and four other lines not shown), independently derived from healthy donors, efficiently lysed 721.221 cells at the E/T ratios shown but were inhibited by target cell expression of either HLA-Cw6 or -Cw7. NK line 986, but not line I, was also inhibited by target cell expression of HLA-G. However, neither of these lines (or the four other lines not shown) was inhibited by target cell expression of HLA-Cw6/G end or -Cw7/G end . HLA-G has a short, 6–amino acid cytoplasmic tail compared with the 33–amino acid cytoplasmic tail of HLA-C. Thus, transfectants were made expressing mutants of HLA-Cw6 in which the cytoplasmic tail was truncated by the insertion of stop codons at the various positions marked in Fig. 1 . NK lines 268 and 264, derived from healthy donors, with phenotypes of inhibition like lines 986 or I in Fig. 2 , were also efficiently inhibited by expression of HLA-Cw6 truncated at positions Lys 315, Ser 325, Ser 332 (not shown), and Ser 335 . Thus, the cytoplasmic tail after residue 314 can be removed from HLA-Cw6 with no effect on its capacity to inhibit NK cell cytotoxicity. To further investigate the sequence of HLA-Cw6 between residues 201 (the Bsu36I cleavage site) and 315 that, when replaced by that of HLA-G, incapacitates the HLA-Cw6 inhibition of NK cell cytotoxicity, a transfectant expressing HLA-Cw6 with the transmembrane sequence of HLA-G (HLA-Cw6/TM G) was made. Similarly, as the most striking difference between the transmembrane sequences of HLA-Cw6 and HLA-G is at position 309, a transfectant expressing HLA-Cw6 with cysteine at position 309 mutated to tryptophan (HLA-Cw6/C309W), the corresponding residue present in HLA-A and -G, was also made. 25/37 (68%) NK lines were inhibited by target cell expression of HLA-Cw6 but efficiently lysed target cells expressing HLA-Cw6/TM G or HLA-Cw6/C309W . Some of the other 12 NK lines inhibited by target cell expression of HLA-Cw6 partially lysed target cells expressing HLA-Cw6/TM G or -Cw6/C309W. Thus, the transmembrane sequence and, in particular, cysteine at position 309 are critical determinants of the capacity of HLA-Cw6 to inhibit the cytotoxicity of many NK cell lines. To investigate which NK cells are sensitive to the transmembrane sequence of HLA-Cw6, many NK clones were examined. Three phenotypes of inhibition are illustrated in Fig. 5 . NK clone 4 was efficiently inhibited by target cell expression of HLA-Cw6, -Cw6/TM G, and -Cw6/C309W . NK clone 2 was efficiently inhibited by expression of HLA-Cw6 but not significantly inhibited by expression of HLA-Cw6/TM G or -Cw6/ C309W . NK clone 82 was partially inhibited by target cell expression of HLA-Cw6 yet was unaffected by target cell expression of HLA-Cw6/TM G or -Cw6/C309W . In total, 33/145 (23%) NK clones selected for their ability to be inhibited by target cell expression of HLA-Cw6 behaved similarly to NK clones 2 or 82 , i.e., they were not inhibited by target cell expression of HLA-Cw6/TM G or -Cw6/C309W. 112/145 (77%) NK clones behaved similarly to NK clone 4 in that they could be efficiently or partially inhibited by target cell expression of HLA-Cw6, -Cw6/TM G, or -Cw6/C309W. One rare NK clone was even costimulated by target cell expression of HLA-Cw6 but not by expression of -Cw6/TM G or -Cw6/C309W (not shown). Also, many of the NK clones found to be inhibited by target cell expression of HLA-Cw7 efficiently lysed target cells expressing HLA-Cw7/G end (not shown), implying that the importance of the transmembrane sequence in NK cell inhibition is not restricted to HLA-Cw6. Some of the NK clones that were inhibited by HLA-Cw6 were phenotyped by mAb staining. All of those tested were CD3 − and CD56 + , as used in the selection, and happened to be GL183 − (mAb for NKIR2) and DX9 − (mAb for NKB1). However, they varied considerably in their expression of NKIR1, being either EB6 bright /HP3E4 bright (clone 84), EB6 dim /HP3E4 dim (clone G), EB6 − /HP3E4 − (clone 57) , or EB6 − /HP3E4 dim (clones 2 and 82; Table I ). Strikingly, clones inhibited by target cell expression of HLA-Cw6, -Cw6/TM G, or -Cw6/C309W were bright for both mAbs EB6 and HP3E4, whereas those inhibited by target cell expression of HLA-Cw6 and not by target cell expression of -Cw6/TM G or -Cw6/C309W were negative or dim for EB6 or HP3E4 (Table I ). Furthermore, only those clones bright for EB6 and HP3E4 were also able to be inhibited by target cell expression of HLA-Cw4, as expected by the specificity of the NKIR1 (data not shown). To directly determine the NK receptors that facilitate inhibition of clones by target cell expression of HLA-Cw6, the effect of blocking NKIR and CD94 with mAb HP3E4 and HP3D9, respectively, was assayed. Two representative clones are shown . NK clone 15 was efficiently inhibited by target cell expression of either HLA-Cw6, -Cw6/TM G, or -Cw6/C309W, and this inhibition could be efficiently blocked by mAb HP3E4 to NKIR1 and not by mAb HP3D9 to CD94. In contrast, NK clone X1 efficiently lysed target cells expressing HLA-Cw6/TM G or -Cw6/C309W, and in this case, the inhibition mediated by expression of HLA-Cw6 was unaffected by mAb to either NKIR1 or CD94. This implies that an NK receptor other than NKIR1 or CD94 mediates inhibition dependent upon the transmembrane sequence of HLA-Cw6. The attachment of the COOH-terminal portion of HLA-G after conserved codon Arg 201 renders the α1 and α2 extracellular domains of HLA-Cw6 or -Cw7 ineffective at inhibition of 25/37 NK cell lines. Thus, cell surface expression of the extracellular domains of an appropriate HLA-C protein is not sufficient for inhibition of NK cell– mediated lysis. Unexpectedly, the transmembrane sequence, and particularly Cys 309, was found to be critical in facilitating HLA-Cw6–mediated inhibition of cytotoxicity of many NK cell lines. 68% (25/37) of NK lines, yet only a smaller subset of NK clones, i.e., 33/145 (23%), were sensitive to the transmembrane sequence of HLA-Cw6. This apparent discrepancy may be because (a) clones are not a proportionate representation of cells within a line, i.e., a selection occurs during the cloning procedure; (b) a selection occurs by the use of NK lines that were inhibited by HLA-Cw6, and the clones used were not necessarily obtained from lines that were inhibited by HLA-Cw6; or (c) a minority of cells within an NK cell line can dominate the overall cytotoxicity of the line through secretion of inhibitory or costimulating cytokines or another method of intercellular communication. All EB6/HP3E4 NKIR1 + NK clones recognized HLA-Cw6 independently of the transmembrane sequence . However, inhibition of EB6 − /HP3E4 − or EB6 dim /HP3E4 dim NK clones by HLA-Cw6 was clearly dependent on the transmembrane region and particularly on Cys 309. This inhibition is unlikely to function through the CD94/NKG2/HLA-E interaction, as there was no correlation between NK lines and clones that were sensitive to the transmembrane sequence of HLA-Cw6 and those for which inhibition could be reversed by anti-CD94 mAb . Moreover, the same leader peptide that facilitates HLA-E expression ( 16 – 18 ) is encoded within each HLA-Cw6 construct. Thus, the NK receptor responsible for recognition dependent upon the transmembrane sequence of HLA-Cw6 is unknown. It may be a previously cloned receptor such as a member of the ILT/LIR family ( 10 – 12 ), or it could be an uncloned receptor such as that postulated to associate with NKIR via zinc ( 38 ). Allogeneic T cell lines that lyse 721.221 transfectants expressing HLA-Cw6 equally lysed target cells expressing -Cw6/TM G or -Cw6/ C309W (data not shown), implying that these receptors do not function on the bulk of T cells. A glycosylphosphatidylinositol (GPI)-linked variant of the mouse class I MHC protein, H-2D d , in H-2 b tumor cells was sufficient to protect against rejection by NK cells after transplantation ( 39 ). Thus, replacement of the transmembrane and cytoplasmic domains of this class I MHC protein with GPI does not affect the capacity of the mouse class I MHC protein to protect against NK cell cytotoxicity. The apparent discrepancy between this finding and the current work may be accounted for in several ways: (a) the GPI linkage and subsequent association of cytoplasmic tyrosine kinases ( 40 ) facilitates signaling within the target cell that may converge with the natural transmembrane-mediated signal pathway; (b) the GPI linkage causes similar cell surface localization/clustering that the cysteine-containing transmembrane sequence facilitates; or (c) the repertoire of inhibitory ligands on mouse NK cells is different from that expressed on human NK cells, and mouse NK cell receptors are less or not at all sensitive to the transmembrane sequence of the inhibitory MHC protein. Aspects of class I MHC protein expression that may be altered by mutation of the transmembrane sequence include: (a) oligomerization ( 41 , 42 ); (b) association with another protein on the target cell surface ( 43 – 46 ); (c) expression of a protein associated in transport; (d) particular localization within cell membrane domains, perhaps facilitated by bound palmitic acid ( 47 , 48 ); (e) conformational change, perhaps induced by association of particular lipids ( 49 ); or (f) capacity for internalization of protein from the cell surface ( 29 ). However, dimerized or oligomeric class I MHC protein at the cell surface was not detected by immunoprecipitating surface-biotinylated class I MHC protein from HLA-Cw6 or -Cw6/TM G transfectants, and no evidence was found of a second specific protein component in the immunoprecipitated material (data not shown). Moreover, confocal fluorescence microscopy failed to reveal any change in the surface or intracellular localization of HLA-Cw6 with an altered transmembrane sequence (data not shown). However, this does not rule out the possibility that the transmembrane sequence is critical in facilitating transient dimerization of HLA-Cw6 or association with another protein upon ligation of an NK receptor. Cysteine at position 309 of class I MHC proteins is most likely located at the interface of the inner leaflet of the lipid bilayer and cytoplasm where the local environment is probably not dissimilar to the extremely viscous surfactant/ aqueous interface widely studied in model colloidal systems such as lipid vesicles and micelles ( 50 , 51 ). The unique physical conditions of the interfacial environment may be critical to the molecular mechanism by which the cysteine facilitates some aspect of class I MHC protein presentation through noncovalent interactions. For example, the transmembrane sequence may control the lateral mobility of class I MHC protein within the cell membrane. In summary, a critical physiological function for the transmembrane sequence of class I MHC proteins in its interaction with a subset of NK cells has been shown. A cysteine within this transmembrane sequence located at or near the interface of the inner leaflet of the lipid bilayer and cytoplasm is particularly important. This cysteine is present in HLA-B and -C proteins but not in HLA-A, -G, -E, -F or CD1a, b, c, or d (although CD1d has a cysteine located centrally within the transmembrane region). HLA-B and -C are major ligands for NKIR, and it is thus intriguing to wonder whether the presence of a cysteine at this location was conserved specifically to facilitate some aspect of NK cell recognition. | Study | biomedical | en | 0.999997 |
10209044 | 6–8-wk-old female (PLSJL/J)F1 mice were purchased from The Jackson Laboratory . Peptides were synthesized on a peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC. Structure was confirmed by amino acid analysis and mass spectroscopy. Peptides used for the experiments were: ENPVVHFFKNIVTPR (MBP p85–99); AASQKRPSQRHG (MBPAc1–11); IGGRVHFFKDISPIA (HPV 7); IGGRVHFFKDISPIS (HPV 13); IGGRVHFFRDISPIG (HPV 40); IGSRVHFFHDISPIT (HPV 32); RKVVTDFFKNIPQRI ( Bacillus subtilis hyp protein X13); and DMTPADALDDRDLEM (HSV VP16). For the peptide treatment a solution of 2 mg/ml of peptide dissolved in PBS, emulsified 1:1 (vol/vol) in IFA was prepared. Mice were injected intradermally with 0.1 ml of the antigen emulsion, twice with a 10-d interval. 10 d after the last injection experimental animals were challenged for EAE. Lyophilized guinea pig spinal cord (gpSCH) was dissolved in PBS to a concentration of 5 mg/ml and emulsified with an equal volume of IFA, supplemented with 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco Labs.). Mice were injected subcutaneously with 0.1 ml of the peptide emulsion, and again on the same day and then 48 h later were injected intravenously with 0.1 ml of a solution of 4 μg/ml Bordetella pertussis toxin in PBS. Experimental animals were scored as follows: 0, no clinical disease; 1, tail weakness or paralysis; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness or paralysis; 5, moribund or dead. Lymph node cells from experimental animals were taken 20 d after challenge for EAE. Cells (5–10 × 10 6 /ml) were incubated in enriched RPMI , supplemented with 1% syngeneic mouse sera with 10 μg/ml peptide for 3 d. After incubation, cells were washed and resuspended for 10 d in enriched RPMI completed with 10% FCS and 10% supernatant of spleen cells activated with concanavalin A (Con A sup). After this period of culture the cells were then activated in the presence of syngeneic irradiated spleen cells (10 7 /ml) and 10 μg/ml peptide for 3 d, washed and incubated for 10 d in enriched RPMI complemented with 10% FCS and 10% Con A sup. The cells were continuously grown in the above conditions for 2-wk cycles. The peptide-specific T cells were used for assays 1 wk after antigen stimulation. T cells (10 4 ) were incubated in 96-well flat-bottomed plates (Corning) with 5 × 10 5 irradiated syngeneic APC in a total volume of 200 μl of enriched RPMI and 10% FCS, and different concentrations of the peptide. After 24 h 100 μl were removed from each well for cytokine secretion analysis in a sandwich ELISA. The remaining cells were incubated for an additional 24 h, pulsed with [ 3 H]thymidine (0.5 μCi of 5 Ci/mmol), harvested, and counted in a beta counter. Peptide binding assays were performed as described elsewhere ( 22 ). In brief, the B cell lymphoma LS102.9 was used as a source of I-A s . The cell line was maintained in vitro by culture in enriched RPMI. Cells were lysed at a concentration of 10 8 cells/ml in PBS containing 1% NP-40, 1 mM PMSF, 5 mM Na-orthovanadate, and 25 mM iodoacetamide. The lysates were cleared of debris and nuclei by centrifugation at 10,000 g for 20 min. Mouse class II molecules were purified as previously described ( 22 ) using the mAb Y3JP (IA b,s -specific), coupled to Sepharose 4B beads. Purified mouse class II molecules (5–500 nM) were incubated with 1–10 nM 125 I-radiolabeled peptides for 48 h in PBS containing 5% DMSO in the presence of a protease inhibitor cocktail. Purified peptides were iodinated using the chloramine-T method. Peptide inhibitors were typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml. The data were then plotted and the dose yielding 50% inhibition (IC 50 ) was measured. Intermediate binding was equivalent to IC 50 in the range of 100–1,000 nM. In appropriate stoichiometric conditions, the IC 50 of an unlabeled test peptide to the purified MHC is a reasonable approximation of the affinity of interaction ( K d ). Peptides were tested in two to four completely independent experiments. Class II peptide complexes were separated from free peptide by gel filtration on TSK2000 columns , and the fraction of bound peptide calculated as previously described ( 22 ). In preliminary experiments, each of the I-A prep was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of class II molecules necessary to bind 10–20% of the total radioactivity. All subsequent inhibition and direct binding assays were then performed using these class II concentrations. TCR antagonism was tested as previously described ( 23 ). In brief, irradiated syngeneic spleen cells were pulsed with a 0.005 μM concentration of MBPp85–99 for 3 h at 37°C. Spleen cells were then washed and used as APCs to the MBPp85–99 (L35) specific T cell line in the presence of different inhibitor concentration. Proliferative responses were measured by [ 3 H]thymidine incorporation. Percentage of inhibition was calculated by the formula described in Table III . We asked whether microbial peptides with structural similarities to the self-peptide MBPp85–99 would block EAE. We made slight modifications of a protocol used previously to prevent EAE by APLs ( 23 ). Mice were injected intradermally twice at 10-d intervals with 0.1 mg of peptide in IFA. 10 d after the last injection the animals were challenged with gpSCH in order to induce EAE. As seen in Table I , the incidence, mean day of onset, and mean peak severity were significantly lower for papilloma virus 40 (VHFFR), papilloma virus 32 (VHFFH), and Bacillus subtilis open reading frame (ORF) (DFFK) than for the IFA alone control or the MBPp85–99/IFA control ( P < 0.001). Mice injected with HPV 7 VHFFK, HPV 13 VHFFK, or the native peptide MBPp85–99 have an increased disease incidence, compared with microbial sequences mutated at the main or secondary TCR contact sites, 91K and 88H respectively, although there was a significant delay of disease onset and mean maximal disease score when compared with IFA control mice ( P < 0.01). Nevertheless, only peptides from HPV 40 and Bacillus subtilis ORF were effective in amelioration of all the clinical parameters, clinical incidence, mean day of onset, and mean peak severity of disease, ( P < 0.001). Thus, viral peptides with limited homology to a self-MBP peptide induce protection to EAE in (PLSJL/J)F1 mice. As seen in Table I , when the motif found in MBPp85–99 is mutated from VHFFK (found in MBP and in HPV 7) to VHFFR in HPV 40, or VHFFH in HPV 32, these microbial peptides reduce EAE induced with gpSCH from 100% incidence (20 out of 20 with IFA alone and 31 out of 40 with MBPp85–99 or HPV 7), to 20% incidence (8 out of 40 with HPV 40 or 32) ( P < 0.01). Thus, a mutation of the major TCR contact site K91 abrogates EAE, a T cell–mediated disease, whereas mutation of the secondary TCR binding site at H88 in Bacillus subtilis ORF totally eliminates disease ( P < 0.001). Since EAE is a T cell–mediated autoimmune disease, these studies support the idea that the microbial sequences mutated at 91K or 88H are APLs: Paul M. Allen, the discoverer of APLs, defines them in this way, “We subsequently defined the term ‘altered peptide ligand' to describe analogues of immunogenic peptides in which the TCR contact sites have been manipulated. While these peptides do not stimulate T cell clonal proliferation, they nevertheless have the capacity to activate some TCR-mediated effector functions. Other peptide analogues do not stimulate any detectable function from the T cells and are simply termed peptide analogues” ( 24 ). Another refinement of the definition in 1998 by Allen again states: “These peptides bind to [MHC class II] with similar affinities, but have different potencies to induce T cell functions: [two substitutions] do not cause T cell proliferation at any concentration, and [one] causes minimal proliferation only at the highest concentrations; all these peptides, however can act as antagonists” ( 25 ). The peptides used in this study are the same: they are APLs that do not cause proliferation of native peptide with VHFFK, but nevertheless antagonize it, and bind with similar affinities to MHC class II (see Table III ). They significantly alter the ability of MBPp87–99 TCR to induce EAE. It is important to note that HPV 7, containing the HFFK motif, cross-reacts with MBPp87–99. Thus an HPV 7 peptide can stimulate MBPp87–99 T cell lines and induce relapsing EAE. Likewise, MBPp87–99 can cross-stimulate an HPV 7 T cell line and induce relapsing EAE ( 1 ). If the K91 is mutated as it is in HPV 32 or 40, EAE is prevented. This dramatic change in T cell phenotype, induction of autoimmune disease versus maintenance of self-tolerance when mutations are made at TCR contact sites, allow these microbial mutant peptides to be called APLs. To analyze the immune responses in the protected animals, T cell lines specific for the viral peptides were generated. After the mice recovered from the acute phase of disease, draining lymph node cells were isolated and restimulated in vitro with either MBP or various viral peptides. Table II shows cytokine profiles of T cell lines isolated from the experimental animals. T cell lines stimulated with HPV 13 and Bacillus subtilis ORF produced both IL-4 and γ-IFN, whereas the T cell line stimulated with MBPp85–99 produced IL-4, but not γ-IFN. T cells stimulated with HSV VP16 peptide, used as a control, lacking the HFFK motif (DMTPADALDDRDLEM), failed to proliferate or produce IL-4 or γ-IFN. These experiments demonstrate that IFA is not critical in the protective effect of these viral peptides, as the T cell lines were derived from animals injected with CFA and antigen. It also indicates that within these animals it is possible to select for lines that can be stimulated by sequences from these viral peptides. Once these lines have been selected, there is no cross-reactivity between the viral peptides and MBP. However, in draining lymph nodes from mice injected with CFA MBPp85–99, T cell responses to MBPp85–99 can be inhibited by viral peptides mutated at the main TCR contact site 91K, but retaining the capacity to bind to MHC class II, I-A s (see Table III below). To study the mechanism of disease protection by the mimicry peptides, T cells isolated from the protected mice and expanded in vitro were reinjected to naive animals and those mice were challenged for EAE induction by gpSCH. As seen in Fig. 1 , the transfer of activated T cells specific for viral peptides from HPV 13 and from Bacillus subtilis ORF and with sequences similar to MBPp87–99 protects from EAE ( P < 0.01 when comparing mean disease score to animals given the T cell line specific for HSV VP16). A Th2 line specific for MBPp87–99 also protects from EAE ( P < 0.01) . We tested the binding of the mimicry microbial peptides to affinity purified I-A s molecules. As shown in Table III , all the peptides tested had intermediate binding to the class II molecules (IC 50 in the 100–1,000 nM range). The possibility that sequence similarity between microbial peptides used in our experiments and the native MBPp85–99 peptide would influence antigen specific responses against the native MBPp85–99 peptide was tested by in vitro inhibition assay. 10 d after immunization of (PLSJL/J)F1 mice with the MBPp85–99 peptide in CFA, draining lymph node cells were tested for the proliferative response to the immunizing peptide alone or together in the presence of mimicry microbial peptides as inhibitors. As observed in Table III , at 1:1 molar ratio the HPV 7 peptide inhibited proliferative responses 62%, HPV 40 peptide inhibits 39%, and Herpes simplex DNA polymerase peptide inhibits 39%. Therefore, cross-reactivity is observed between the native MBPp85–99 peptide and the mimicry microbial peptides at the level of inhibition of proliferative responses. Inhibition of proliferative responses may have been due to the capacity of the microbial peptides to mimic native MBP, and to bind to the MHC class II I-A s molecules, partially inhibiting presentation of the MBPp85-99 to specific T cells. To rule out a bystander effect by the microbial mimicry peptides, we compare the protective effect in EAE induced by either gpSCH or MBPpAc1–11. In Fig. 2 , we show that the Bacillus subtilis peptide injected into IFA inhibits induction of EAE induced by gpSCH, but does not inhibit disease induced by MBPpAc1–11. Therefore, the regulatory effect of mimicry peptides requires the presence of the MBPp85–99 epitope in the EAE-inducing antigen. We tested for TCR antagonism using an assay by Karin et al. ( 23 ). In this assay we inhibit the proliferative response to wild-type MBPp85–99 by prepulsing irradiated splenic APCs with 0.005 mM of MBPp85–99, then washing and using these APCs to stimulate an MBPp85–99–specific T cell line in the presence of a putative antagonist. The peptide HPV 40 (containing the motif VHFFR) inhibited proliferation by 40%, whereas a control peptide lacking the HFFK motif did not inhibit proliferation at all . “In MS—as in other autoimmune diseases—there has been feverish debate between those who believe that the disease is triggered by an environmental agent and those who champion a genetic basis. But there is actually a reasonable reconciliation of these opposing views: certain genes conferring susceptibility to the disease and certain factors in the environment are both critical for the development of autoimmunity, and this is particularly true for MS” ( 7 ). Molecular mimicry provides a scheme whereby viral sensitization in the blood leads to activation of T cells ( 26 ). These enter the brain where they encounter their cognate mimic in myelin. We have detected a number of microbes whose amino acid sequences can activate anti-myelin T cells from MS patients, as well as bind to anti-myelin antibodies eluted from MS brain material ( 19 , 21 ). Molecular mimicry also allows for reconciliation of the genes versus the environment debate: genomic searches for genes linked to MS susceptibility reveal that the most important gene in determining susceptibility to MS is HLA ( 28 – 30 ). HLA is of course critical for selecting the appropriate mimic and presenting it to the immune system. Moreover, many different viruses mimic various parts of the myelin sheath, so inflammation in the white matter of the brain may ensue from an immune response to a variety of microbes. Thus, the hope of finding the virus that triggers MS may remain elusive forever ( 7 ). Our study shows how molecular mimics may modulate autoimmune disease. Earlier work by Gautam et al. ( 31 ) had demonstrated that a polyalanine peptide with only five native MBP residues is able to induce EAE in (PLSJL/J)F1 mice. Further analysis also showed that an 11-amino acid peptide, consisting mostly of alanines with only four native Ac1–11 residues, was able to induce T cell hybridoma proliferation. Taking an approach of introducing either d-amino acids or unnatural amino acids in place of l-amino acids into MBPpAc1–11 analogues, we showed that T cells recognize only a short stretch of six or seven amino acids. More importantly, this stretch contains only four native MBPpAc1–11 residues. We also tested T cell recognition in vivo, using EAE as a measure of activation. We show that a short peptide of six amino acids with a core of only five native Ac1–11 amino acids induces EAE ( 31 , 32 ). A herpes virus Saimiri (HVS) peptide, A A Q R RPS RPFA, with a limited homology to MBP1–11 peptide, A S Q K RPS QRHG, (bold letters show homology) can stimulate a panel of MBP1–11–specific T cell hybridomas, and more importantly cause EAE in mice. We demonstrate that this is due to cross recognition of these two peptides by TCRs. This HSV peptide with homology at just five amino acids with a self-peptide can induce clinical signs and histologic evidence of EAE in mice ( 33 ). Relapsing EAE has been induced with two peptides bearing the HFFK motif containing the primary TCR and MHC contact for I-E in the Lewis rat, I-A in the SJL mouse, and DRB1*1501 in humans ( 5 , 17 , 18 , 23 ). Using a passive transfer protocol, T cells specific for an HPV 7 peptide (IGGRVHFFKDISPIASSE) were found to induce relapsing EAE. These T cells could be activated by MBPp87–99 to induce EAE, and MBPp87–99 T cells could also be stimulated by the HPV 7 peptide to induce EAE. Active EAE with this papilloma peptide was also induced. Another viral peptide from EBV (RAHPVYFFKSACPPA) could activate the papilloma virus–specific T cells and induce EAE by passive transfer ( 1 ). These results have practical significance for the success of APL therapy in MS patients. The APLs now in Phase II clinical trials in MS ( 2 ) have a K→ A substitution at position 91 and thus, neither bind anti-MBP antibody nor trigger MBP-specific T cells. Administration of soluble native versions of myelin antigens may have dangerous consequences. Genain et al. showed that EAE induced in marmosets by immunization with myelin oligodendroglial glycoprotein (MOG) could be delayed by intraperitoneal treatment with soluble MOG; however, treated animals developed a severe late form of the disease ( 34 ). In these animals, MOG-specific T cell proliferative responses were transiently suppressed, cytokine profiles were shifted from a Th1- to a Th2-type pattern, titers of autoantibodies to MOG were enhanced, and autoimmune disease was exacerbated ( 34 ). This implies that provoking a vigorous anti-myelin reaction with a native peptide could have dangerous consequences in a clinical setting ( 35 ). APLs work in part by altering cytokine production in T cells that respond to self-antigens ( 36 – 40 ). For example, in the Lewis rat administration of MBPp87–99 (K91→ A), an APL altered at the primary TCR contact residue K91, reversed paralysis in EAE, and reduced production of the proinflammatory cytokine TNF-α ( 23 ). Another APL, MBPp87–99 (96P→ A), reversed paralysis in EAE and increased production of IL-4 at the site of disease ( 5 ). The effect of this APL was reversed by the in vivo administration of anti–IL-4 antibody ( 5 ). In the studies presented here, T cell lines with specificity for viral sequences that resemble MBPp85–99, but were not identical to MBPp85–99, produced IL-4 and γ-IFN, two cytokines known to suppress EAE ( 5 , 41 – 45 ). Despite the fact that systemic administration of γ-IFN is protective in EAE, γ-IFN can induce MHC class II on astrocytes ( 46 ), and allow these glial cells to present myelin antigens to encephalitogenic T cells. Although in EAE systemic administration of γ-IFN is protective, in MS administration of γ-IFN provokes exacerbation of disease ( 47 ). Interestingly, a T cell line specific for MBP that produced IL-4 was also able to suppress EAE . Both IL-4 and γ-IFN are capable of suppressing EAE. Thus, lines specific for microbial sequences like HPV 13 or Bacillus subtilis ORF produce both IL-4 and γ-IFN and suppress EAE, whereas a T cell line specific for MBP producing IL-4 and no γ-IFN also protects. From previously published work we know that antibody to γ-IFN and antibody to IL-4 both exacerbate EAE ( 5 , 48 ). Thus, the production of antiinflammatory cytokines like IL-4 by T cells responding to microbes, whose sequences resemble but are not identical to the self-epitope MBPp87–99, has potent effects on in vivo disease. These T cell lines can inhibit EAE in (PLSJL/J)F1 animals, induced by gpSCH. This homogenate contains epitopes such as MBPpAc1–11, and MBPp35–47, as well as PLP epitopes, that dominate the pathogenic response (8– 10, 13). The trans-acting effect of such T cells, producing antiinflammatory cytokines, is thus able to shut down a diversity of immune responses ( 2 ). Suppression of autoimmune disease with microbial sequences can also be achieved by naked DNA immunization with minigenes encoding the core motif of MBPp85–99 ( 48a ). Thus, microbial genomes may direct immunization via the information encoded in the DNA itself, and this immunization might include sensitization to altered peptides of self ( 49 , 50 ). The notion that microbial sequences can act as APLs and suppress autoimmune disease appears to be novel. Combined with the observation that microbial mimics can also induce EAE ( 1 , 33 , 51 ), the concept of molecular mimicry provides a framework for explaining the modulation of immune responses to self, both in the development of autoimmune disease and in protection from autoimmunity. A recent report by Zhao et al. demonstrated that a coat protein of HSV type 1 could be recognized by autoreactive T cells that target corneal antigens in a murine model of autoimmune herpes stromal keratitis ( 52 ). Mutant HSV type 1 viruses that lacked this epitope did not induce autoimmune disease. Our report reveals that mutant viruses can block autoimmunity, and this appears to be a novel observation. The interaction of the immune system with microbes may allow the selection of viral subtypes. It is interesting to speculate that attenuation of the immune response by a peptide derived from a papilloma viral subtype, containing an APL-like motif, may be desirable for viral survival. A virus that can subvert immunity might be selected because it could survive and persist, instead of being eradicated in the wake of an autoimmune response. The degeneracy of T cell recognition of MBPp87–99 has been clearly shown with combinatorial peptide libraries ( 53 ). At least in the case of T cells specific for MBPp87–99, there may be a delicate physiological interplay between self- and microbial antigens, allowing the modulation of autoimmune disease and the persistence and survival of mutant microbes. Attenuating inflammation in the brain may allow microbes to survive, sequestered within the central nervous system. It is remarkable that certain viral subtypes are mutated exactly at a main TCR contact site, and such mutations may represent an adaptive response of a virus, which then acts as an APL. | Study | biomedical | en | 0.999997 |
10209045 | Specific pathogen-free C3H/HeJ, Balb/c, and C57Bl/6 male mice were purchased from the National Cancer Institute– Frederick Cancer Research Facility Animal Production Area. C57Bl/6 gld/gld and C57Bl/6 lpr/lpr male mice were purchased from The Jackson Laboratory . C3H/HeJ gld/gld male mice were generated from a breeder colony maintained in our facility and used between 8 and 14 wk of age. Mice were housed in a pathogen-free barrier facility accredited by the American Association for Accreditation of Laboratory Animal Care, in accordance with current U.S. Department of Agriculture, Department of Health and Human Services, and National Institutes of Health regulations and standards. All animal procedures were approved by the Institutional Animal Care and Use Committee. A bank of six Westinghouse FS40 sunlamps was used as a source of UV radiation as described ( 25 ). DTH responses were assessed as previously described ( 9 ). In brief, mice were shaved and exposed to UV-B radiation (2–5 and 15 kJ/m 2 for C . albicans and alloantigen, respectively). 3 d later, mice were sensitized by subcutaneous injection of antigen (10 7 formalin-fixed C . albicans or 5 × 10 7 Balb/c spleen cells or cell equivalents). 6–10 d after antigen sensitization, mice were challenged by injecting either purified C . albicans protein (Allercheck, Inc.) or 10 7 Balb/c spleen cells in the footpad. 24 h later, footpad swelling was quantitated using a spring loaded micrometer (Swiss Precision Instruments). Specific footpad swelling (Δswelling) was determined by subtracting the footpad swelling in mice that were challenged but not sensitized from that observed in mice that were sensitized and challenged. Percent suppression was calculated as: % suppression = 1 − ( T − N / P − N ) × 100, where N = negative control (response of unsensitized mice to challenge), P = positive control (response of sensitized mice to challenge), and T = test group (response of mice given UV irradiation before sensitization and challenge). Treatment groups consisted of 3–6 (typically 5) mice; both hind footpads were measured. CHS responses were determined as previously described ( 43 ). In brief, for FITC responses, the abdominal hair of mice was shaved, their ears protected with electrical tape, and the animals exposed to UV-B radiation (2 kJ/m 2 ). 3 d later, the dorsal hair was shaved and the animals sensitized by epicutaneous application of 400 μl of 0.5% FITC (Isomer I, Aldrich Chemical Co. ) in acetone–dibutylphthalate (1:1, vol/vol). 5–7 d later, the mice were challenged by applying either 10 μl 0.5% FITC to the ventral and dorsal surfaces of both ears. Ear swelling (Δswelling) was quantitated 24 h later using a spring loaded micrometer and specific ear swelling determined by subtracting the ear swelling in mice challenged but not sensitized from that observed in mice that had been sensitized and challenged; percent suppression was calculated as described for DTH responses. For transfer of splenic suppressor cell populations, mice were killed, spleens harvested, and single cell suspensions prepared immediately following DTH or CHS analysis. Approximately 10 8 spleen cells were injected into the tail veins of NR, naive recipient mice and the animals immediately sensitized by subcutaneous injection (10 7 formalin-fixed C . albicans or 5 × 10 7 Balb/c spleen cells) or epicutaneous application (400 μl 0.5% FITC). 6–10 d later, mice were challenged as described above and DTH or CHS responses determined 24 h later. C3H/HeJ mice were shaved and exposed to 15 kJ/m 2 UV radiation as described above. 3 d after UVR, mice were killed and inguinal, axillary, and brachial lymph nodes harvested. Lymph nodes were mechanically dissociated and washed, and RNA was extracted with Trizol ( GIBCO BRL ) per manufacturer's instructions. Reverse transcriptase (RT) and PCR reactions were performed with the GeneAmp PCR kit ( Perkin-Elmer Corp. ) using the following primer sequences: FasL, 5′-ATCCCTCTGGAATGGGAAGA-3′ (forward), 5′-CCATATCTGTCCAGTAGTGC-3′ (reverse); β actin, 5′-TCCTGTGGCATCCATGAAACT-3′ (forward), 5′-CTTCGTGAACGCCACGTGCTA-3′ (reverse). 35 cycles of PCR were performed: 30 s at 94°C, 45 s at 55°C, and 60 s at 72°C, using a Perkin-Elmer Gene Amp 9600. For DTH and CHS analysis, the probability of no difference between treatment and controls was analyzed in a factorial ANOVA using Fisher's protected least significant difference test with a 5% significance level. Statistical analyses were performed with Statview software (Abacus Concepts; v4.5). Normal mice exposed to a single dose of UVR before immunization at an NR site with formalin-fixed C. albicans or FITC exhibit a profound suppression of DTH or CHS response, respectively. To address the potential role of Fas/FasL interactions in UVR-induced systemic immune suppression, lpr or gld mice were evaluated for UV-induced immune suppression of CHS and DTH responses. The lpr mutation encodes an abnormal Fas gene containing an early retroviral transposon insertion that results in premature termination ( 44 – 47 ). Low levels of Fas expression (up to 50% of the level observed in wild-type mice) have been reported in lpr mice, demonstrating an incomplete Fas loss in these animals ( 46 , 48 ). Mice harboring the gld mutation have a complete loss of biologically active FasL as a result of a point mutation in the FasL gene ( 49 – 51 ). The requirement for Fas/FasL interactions in UVR-induced immune suppression was first evaluated by comparing responses in wild-type C3H/HeJ (C3H) and FasL-deficient C3H/HeJ gld/gld (C3H/ gld ) mice. Both groups of animals were exposed to a single dose of UVR and immunized 3 d later at a distant NR site by epicutaneous application of FITC or subcutaneous injection of C . albicans ( 9 ). 6–10 d later, mice were challenged either on the pinnae or in the footpad with the sensitizing antigen to elicit CHS and DTH responses, respectively. Representative results from one such experiment are shown in Fig. 1 . In wild-type C3H mice, UVR exposure potently suppressed both CHS and DTH responses compared with NR, positive control mice . In direct contrast, UVR-exposed C3H/ gld mice exhibited no such immune suppression. Similar results were observed in C57Bl/6 (B6) and C57Bl/6 gld/gld (B6/ gld ) mice for DTH to C . albicans (data not shown). Thus, when FasL is nonfunctional, UVR-induced immune suppression is abrogated. To explore the effects of diminished Fas function on UVR-induced immune suppression, we next examined the DTH response of wild-type B6 and Fas-deficient C57Bl/6 lpr/lpr (B6/ lpr ) mice acutely exposed to a single dose of UVR and immunized with C . albicans . Results from a representative experiment are shown in Fig. 1 . Unlike mice containing the gld mutation, DTH induction was significantly suppressed in both UVR-treated B6/ lpr and wild-type B6 mice compared with NR control mice in each group. Notably, UV-induced suppression in lpr mice was only half that observed in wild-type B6 mice with intact Fas expression (40% suppression in lpr mice compared with 83% in wild-type mice). The observation that B6/ lpr mice have intermediate sensitivity to UVR-induced immunosuppression is not likely the result of differences in genetic background between the two mouse strains, as B6/ gld mice also exhibited a complete absence of UVR-induced immune suppression (data not shown). Instead, these findings likely reflect the leaky nature of the lpr mutation and, moreover, suggest that the induction of Fas after UV exposure may reestablish the capacity to induce systemic immune suppression. Indeed, Fas upregulation has been recently reported to occur in lpr mice exposed to gamma irradiation ( 52 ). Taken together, these results point toward a critical requirement for Fas/FasL interactions in primary, UVR-induced systemic suppression of CHS and DTH induction. Earlier work in our laboratory demonstrated the presence of DNA damage (cyclobutane pyrimidine dimers) in skin-derived dendritic cells up to 1 wk after a single acute exposure of UVR ( 27 ). As DNA damage can induce FasL expression, a potential mechanism for the requirement for FasL in UV-mediated suppression might be the inappropriate expression of FasL in the draining lymph nodes after UVR. To explore this possibility, the skin-draining lymph nodes from wild-type mice were removed 3 d after UVR and FasL expression determined by RT-PCR. As shown in Fig. 2 , FasL mRNA was markedly induced in the lymph nodes of mice that received 15 kJ/m 2 UVR but was undetectable in untreated control animals. Specificity was demonstrated using L929 murine fibroblasts and unactivated lpr/ lpr splenocytes as negative and positive controls for FasL expression, respectively ( 53 ). FasL induction in the skin-draining lymph nodes after UVR points to an interrelationship with DNA damage and suggests a potential scenario in which inappropriate FasL expression eliminates antigen- responsive T cells ( 54 ) or serves to clonally expand a suppressor cell population ( 55 ). Experiments are currently underway to test these possibilities. UVR-induced suppression of the DTH response to whole alloantigen differs from that of FITC or C . albicans in that it is mediated by photoisomerization of cis-urocanic acid and is UV-induced DNA damage–independent (Kripke, M.L., unpublished observations). Such findings suggest that UVR-induced suppression of the DTH response to alloantigen differs mechanistically from that of C . albicans . To evaluate the role of FasL in UVR-induced immune suppression of DTH responses to whole alloantigen, C3H, C3H/ gld , B6, and B6/ gld mice were exposed to a single dose of UVR and immunized with intact Balb/c spleen cells, and DTH responses were measured. In three independent experiments, UVR exposure potently suppressed DTH responses in both strains of FasL-deficient gld mice at levels comparable to those of matched, wild-type control mice. These results demonstrate that, in contrast to other antigen systems tested (FITC and C . albicans ), primary UVR-induced suppression of DTH to whole alloantigen can proceed in a manner independent of host-derived FasL. To resolve the differences in FITC, C . albicans , and alloantigen requirements for FasL in the mediation of UVR-induced immune suppression, a more detailed analysis of alloimmunization was undertaken. Whereas FITC and formalin-fixed C . albicans require processing and presentation by host APC, whole allogeneic splenocytes may bypass such a requirement through direct antigen presentation on the various cell populations in the sensitizing inoculum. To this end, we queried whether FasL was required for UVR- induced suppression when allogeneic splenocytes were disrupted before immunization. UVR-induced systemic suppression of responses to intact and disrupted allogeneic spleen cells was compared in C3H and C3H/ gld mice by exposing mice to a single dose of UVR, immunizing 3 d later with either intact or disrupted allogeneic Balb/c spleen cells, and challenging 6–10 d later with intact spleen cells to elicit a DTH response. Disrupted allogeneic spleen cells were prepared by several freeze–thaw cycles and sonication to insure uniform dispersion. Results from one of two consistent experiments are shown in Fig. 4 . DTH responses were suppressed in UVR-treated wild-type mice whether intact or disrupted alloantigen was used for sensitization . In contrast to the potent suppression of DTH observed in UVR-treated C3H/ gld mice immunized with intact allogeneic spleen cells , UVR-treated C3H/ gld mice immunized with disrupted allogeneic spleen cells failed to demonstrate such suppression. The lack of primary, UV-induced immunosuppression in FasL-deficient mice immunized with disrupted alloantigen is in accordance with our findings using FITC and C . albicans , suggesting that such suppression is dependent upon FasL and host-derived APC for antigen presentation. Antigen-specific T suppressor cells capable of transferring antigen-specific suppression to a naive host exist in the spleens of mice exposed to UVR and sensitized to antigen ( 11 – 15 ). Although primary, UVR-mediated suppression was absent in FasL-deficient mice, it remained uncertain whether transferable suppressor cell function was also absent in such mice. To address this premise, we compared suppressor cell activity in adoptively transferred spleen cells from immunized, UVR-exposed wild-type and gld donors. These experiments were carried out by exposing C3H and C3H/ gld mice to a single dose of UVR followed by immunization and challenge with FITC, C . albicans , or alloantigen. On the day that CHS and DTH responses were measured, spleens from UVR-exposed animals and NR control animals were harvested and transferred into naive recipient mice. Recipient mice were immediately sensitized with the indicated antigen and received antigenic challenge 6–10 d later. Mice receiving NR, antigen-primed spleen cells from either C3H or C3H/ gld donors showed potent CHS and DTH responses to FITC and C . albicans , respectively. Animals that received spleen cells from UVR-exposed, antigen-primed C3H mice demonstrated a suppression of both CHS and DTH responses, confirming the presence of UV-induced splenic suppressor cells as expected . In contrast, adoptively transferred UVR-exposed spleen cells from C3H/ gld mice primed with either FITC or C . albicans failed to inhibit antigen sensitization in naive mice. Although these experiments suggest a loss of transferable suppressor cell activity in the absence of donor-derived FasL, they are not definitive, as such mice also lack primary UVR-mediated suppression, which may be essential for the subsequent generation of transferable suppressor cells. To this end, transferable suppression was also evaluated in C3H/ gld mice immunized with allogeneic spleen cells in which primary, UVR-mediated immune suppression was intact. For these experiments, spleen cells from UVR-exposed, FasL-deficient C3H/ gld (data not shown) or B6/ gld mice immunized with allogeneic spleen cells were transferred to naive, matched wild-type recipients . As expected, spleen cells from wild-type mice that received UVR before antigen priming showed potent suppressor cell activity when transferred into matched wild-type recipients . Consistent with our previous experiments, spleen cells from FasL-deficient mice that received UVR before antigen priming showed no such transferable suppressor cell activity. That C3H/ gld and B6/ gld mice exhibited potent UVR-induced primary suppression but not splenic suppressor cell activity when immunized with allogeneic whole spleen cells suggests that these events are mechanistically dissimilar and that transferable suppression is strictly dependent upon FasL expression on the donor population. In further support of this premise, wild-type mice that received UVR before immunization with disrupted allogeneic spleen cells exhibited transferable suppressor cell activity comparable to that of C3H mice immunized with intact allogeneic spleen cells. Spleen cells from FasL-deficient mice that received UVR before immunization with disrupted alloantigen, however, again demonstrated no such transferable suppressor cell activity (data not shown). These results substantiate that donor-derived FasL expression is uniformly required for the generation and/or effector activity of UV-induced transferable suppressor cells for all antigen systems tested. In the experimental models above, we have shown a stringent requirement for donor-derived FasL in UV-mediated transferable suppression. To discern whether FasL was also required in recipient mice, we evaluated UV-induced transferable suppression in FasL-deficient recipients. Such experiments were carried out by transferring spleen cells from wild-type mice that received UVR before immunization with C . albicans into either wild-type or FasL-deficient recipient mice (C3H or C3H/ gld ). Results from one such experiment are shown in Fig. 6 . Consistent with our previous findings , spleen cells from UVR- exposed, C . albicans –immunized C3H donor mice suppressed subsequent antigen responsiveness in naive C3H recipients . Similarly, UVR-exposed, C . albicans –immunized C3H donor spleen cells markedly suppressed subsequent antigen responsiveness in C3H/ gld recipients , ruling out a requirement for recipient-derived FasL in transferable suppression induced by UVR. No suppression was observed when NR, C . albicans –immunized C3H splenocytes were transferred to either C3H or C3H/ gld recipients, confirming a requirement for both UVR and antigen exposure in the generation of suppressor cell activity ( 16 , 17 ). Fas and FasL are complementary receptor–ligand proteins that induce apoptosis in many cell types ( 30 ). Fas is constitutively expressed in numerous tissues ( 56 ) and is rapidly upregulated in activated lymphocytes ( 31 , 32 ). Although constitutive FasL expression is restricted to only a few tissues ( 57 ), transient FasL upregulation has been observed in a variety of cell types after genotoxic damage or cellular injury ( 3 , 36 , 58 ). Activated Fas + lymphocytes can upregulate FasL upon T cell receptor engagement ( 39 – 41 ). Fas/FasL-induced apoptosis has been shown to play a critical role in the control of lymphocyte proliferation, peripheral tolerance, and specific immune responses occurring in discrete organ environments ( 35 , 59 ). The effect of UVR on immune function shares several commonalities with Fas/ FasL-driven immunoregulation. UVR ( 6 , 10 ), like FasL ( 35 , 37 ), can mediate antigen-specific immune suppression. Moreover, UVR ( 19 – 23 ), like FasL ( 42 ), has been shown to shift the activation of T cells from a Th1- to a Th2-type immune response. Therefore, we were prompted to examine the role of Fas/FasL interactions in UVR-induced immune suppression. Our observations are important in that they identify FasL as a fundamental constituent of both primary and transferable antigen-specific suppression induced by UVR (Table I ). The central role of FasL in UV-induced systemic suppression of CHS and DTH responses varies from that of Th2-like immunomodulatory cytokines and cis-urocanic acid, which are not shared conjointly in the suppression of CHS and DTH responses (19–21; Kripke, M.L., unpublished observations). For example, IL-10 appears to be essential for systemic UVR-induced suppression of DTH responses ( 20 ), whereas TNF-α is essential for CHS suppression ( 19 , 60 ). The selective requirement of FasL for UVR- mediated suppression of CHS responses to FITC and DTH responses to Candida , but not alloantigen, is reminiscent of our previous finding that repair of UV-induced DNA damage could restore immune responses to FITC and Candida but not alloantigen (25–27; Kripke, M.L., unpublished observations). Taken with the recent report that DNA damage can activate the FasL promoter and upregulate FasL expression ( 61 ), our findings raise the interesting possibility that UVR-induced DNA damage and FasL are interrelated in the induction of UVR-induced immune suppression. The potent induction of FasL mRNA in skin-draining lymph nodes after UVR lends additional credence to this premise . How might DNA damage, FasL, and immunomodulatory cytokines interrelate in the induction of UVR-induced immune suppression? First, DNA damage by a variety of agents, including UV light, has been shown to induce the expression of both Fas and FasL ( 3 , 36 , 61 ). Our laboratory has previously demonstrated that cyclobutane pyrimidine dimer–containing APC are present in the lymph nodes of UVR-exposed mice ( 27 ). Such APC may upregulate FasL and eliminate responding Fas + T cells as has been recently reported for dendritic cells in vitro ( 42 , 54 ). Second, aberrant FasL upregulation coupled with alterations in Fas sensitivity as a result of UVR-induced immunomodulatory cytokine production may contribute to the termination of immune responses by inappropriate apoptosis of T cells and/or APC ( 38 , 40 , 41 ). Third, suppressor T cells responding to antigen in the context of the UVR cytokine milieu may differentiate along a novel pathway requiring FasL as a growth factor ( 55 , 62 ). Published observations, along with this report, collectively favor a model in which UV-induced changes in APC phenotype/function are pivotal in the induction of antigen-specific immune suppression. Our data involving intact and disrupted alloantigen point toward a requirement for FasL on UVR-exposed host APC. While such APC are required for the antigen presentation of FITC, C . albicans , and disrupted alloantigen, intact allogeneic spleen cells may circumvent this requirement by providing a source of FasL while acting as their own APC. These results suggest that the nature of the antigen (and thus the APC involved) critically determines the requirement for host-derived FasL in primary UVR-induced immune suppression. What then might be the requirement for FasL in the generation and activity of transferable suppressor cells? Considering the low penetrance of UVR in the skin, it appears unlikely that direct UVR exposure and UV-induced DNA damage occurs on T cells. It is conceivable, however, that DNA-damaged APC may influence the development of such T cells. In this regard, we have previously shown that DNA-damaged APC cluster with suppressor T cells in the draining lymph nodes after UVR and antigen exposure ( 27 , 43 , 63 ). Taken with the observation that Fas ligation can induce proliferation in some T cells ( 62 ), it is possible that FasL on DNA-damaged APC may act as a growth factor for UV-induced T suppressor cells in the context of the UVR-treated animal. Interestingly, Groux et al. ( 64 ) have recently described an IL-10 driven, antigen-specific CD4 + T cell that can potently suppress antigen-specific immune responses in vivo. Such findings suggest that UVR-induced immunoregulatory Th2 cytokines such as IL-10 ( 18 – 20 ) may also participate in the differentiation and maintenance of the suppressor cell population. On the other hand, FasL may be required for effector activity of the UV-induced suppressor cells, perhaps by inducing apoptosis in the responding recipient T cell population. Experiments are currently in progress to test these possibilities. Recent studies highlight the complexity of the immunomodulatory effects of UV in vivo. Hart et al. have documented mast cell–derived histamine as a component of the UV-induced systemic immunosuppression of DTH responses to alloantigen ( 65 ). In contrast to our studies on UV-induced systemic immune suppression, Schwartz et al. have shown a nonessential role for FasL in UV-induced local immune suppression ( 66 ). The local model of UV-induced immune suppression differs markedly from the systemic model in both the route of administration (antigen is administered through the UV-irradiated site), specific cytokine involvement, and the requirement for FasL. For example, UVR-induced suppression of local responses is TNF-α dependent and IL-10 independent and involves the production of cis-urocanic acid ( 67 ). In contrast, UVR-induced systemic immune suppression is independent of both TNF-α and cis-urocanic acid but dependent upon IL-10 production (19, 20; Kripke, M.L., unpublished observations). Collectively, these findings emphasize mechanistic differences between UVR-mediated local and systemic suppression and suggest the existence of at least two nonoverlapping pathways in the generation of systemic UVR-induced immune suppression. One pathway requires FasL on host-derived APC and is sensitive to reversal by the repair of UV-induced DNA damage (25–27; Kripke, M.L., unpublished observations); the other requires histamine ( 65 ) and is independent of host-derived FasL. Interestingly, both pathways require FasL for the generation of transferable suppression but may differ in their requirement for FasL in the host ( 66 ). In summary, our experiments document that Fas/FasL interactions are essential for UVR-induced systemic suppression of CHS and DTH responses to antigens presented by host-derived APC (Table I ). The requirement for Fas/ FasL in UVR-induced immune suppression can be eliminated if antigen presentation bypasses the requirement for host-derived APC (intact alloantigen). Moreover, host- derived, but not recipient-derived, FasL expression is critically required for the generation and/or function of UVR-induced suppressor cells. The crucial role of FasL in both systemic primary and transferable UV-induced immune suppression suggests that the dysregulation of Fas-mediated apoptosis may ultimately underlie both processes. | Study | biomedical | en | 0.999997 |
10209046 | Ig transgenic lines of C.B-17 scid mice hemizygous for the H chain tgs, M54 ( 24 ), 3H9 ( 25 ), or the kappa L chain tg, Vκ8 ( 26 ), have been described previously ( 20 ). M54/Vκ8 and 3H9/Vκ8 scid mice were obtained by crossing individual tg lines and typing offspring for the presence of the respective tgs. Ig transgenic control mice, heterozygous for the scid mutation (denoted as scid/+ or s/+ mice), were obtained by crossing the above transgenic scid lines with C.B-17 wild-type mice. Mice used in this study were between 8 and 12 wk of age. Bone marrow cells were flushed from femurs with staining medium using a syringe and 22 gauge needle. The cells were then dispersed by gentle pipetting, treated with 0.165 M NH 4 CL, washed, and resuspended in staining medium and passed through a sterile nylon screen. B cell hybridomas were obtained by fusing unstimulated splenic cells from adult M54/Vκ8 (or 3H9/Vκ8) scid and scid/+ mice in a manner previously described ( 27 ) using Ag8.563 ( 28 ) as the cell fusion partner. Bone marrow cell suspensions were analyzed for the presence of B lineage cells representing different stages of development ( 17 ). In brief, cell suspensions were stained with Cy5 (Biological Detection Systems, Inc.) or allophycocyanin (APC; PharMingen ) conjugated anti–CD45(B220), FITC-conjugated anti–CD43, or biotinylated anti–IgM. Binding of biotinylated antibodies was revealed by Texas red conjugated Streptavidin (Southern Biotechnology). B220 + CD43 + IgM − , B220 + CD43 − IgM − , and B220 + CD43 − IgM + cells were enumerated or sorted by multiparameter flow cytometry using a dual laser FACStar Plus ® ( Becton Dickinson & Co.). Forward and light-angle scatter gates were set to include lymphoid cells only. Dead cells were identified by propidium iodide staining and excluded from analysis. To distinguish early B lineage subsets, B220 + CD43 + -gated cells were stained with phycoerythrin-conjugated anti–BP1 ( 29 ) and biotinylated anti–heat stable antigen (HSA) ( 17 ) (both reagents were provided by R. Hardy, Fox Chase Cancer Center, Philadelphia, PA). Genomic DNA was prepared from sorted cell subsets (0.5–1.0 × 10 6 cells) as described previously ( 30 ) and dissolved in water at a concentration corresponding to 10 5 cell genome equivalents/μl. Ligation-mediated PCR (LM-PCR) ( 31 – 33 ) was used to assay DNA samples for double strand breaks (DSBs) resulting from the initiation of H chain gene rearrangement. Initiation of V(D)J rearrangement results in site-specific DSBs at the recombination signal/coding borders of V, D, and J elements: two kinds of broken DNA molecules are generated; covalently closed (hairpin) coding ends and blunt signal ends ( 34 , 35 ). We assayed for broken molecules with signal ends; specifically, those with JH signal ends and those with 5′ or 3′ DHfl16.1 (DHfl) signal ends. We also assayed for signal joints (by inverse PCR), completed DH-to-JH rearrangements and unrearranged JH loci as scored by the retention of germline sequence immediately upstream of JH1. Assays were performed as follows. A double strand linker was ligated to DNA (equivalent to ∼4 × 10 5 cell genomes). The linker was constructed according to Roth et al. ( 32 ) by annealing two oligonucleotides, DR19 (5′-CACGATTCCC-3′) and DR20 (5′-GCTATGTACTACCCGGGAATTCGTG-3′). After ligation, different dilutions of the ligation reaction (input DNA) were used to perform PCR amplifications of one or more of the following: (a) linkered JH signal ends using DR20 and an oligonucleotide (MB221) complementary to a sequence immediately 5′ of JH1 (5′-TCTCTTGTCACAGGTCTCACTATGC-3′); (b) linkered 5′ DHfl signal ends using DR20 and an oligonucleotide (MB222) complementary to a sequence 5′ of DHfl (5′-GCCTTCCACAAGAGGAGAAG-3′); (c) linkered 3′ DHfl signal ends using DR20 and an oligonucleotide (MB241) complementary to a sequence 3′ of DHfl (5′-TGGGTCAGTGGTCAAGACTCG-3′); (d) signal joints resulting from the joining of JH1, JH2, or JH3 signals to the 3′ DHfl signal using MB221 and MB241; (e) DJH coding joints using a DHfl/DHsp primer MB109 (5′-CCGAATTCGTCCTCCAGAAACAGACC-3′) and a primer (MB 92) complementary to a JH4 sequence (5′-GCCGGATCCCTTGACCCCAGTAGTCC-3′); (f) retained sequence immediately upstream of JH1 using MB221 and MB92; and (g) α actin sequence using actin-specific primers ( 17 ). The level of amplified α actin product served as a control for the amount of input DNA. DNA was amplified in a 50-μl reaction volume containing each primer at a concentration of 0.5 μM, 200 μM MgCl 2 , 10 mM Tris-HCL, pH 8.3 at 25°C, 50 mM KCL, 0.001% gelatin, 200 μM (each) dTTP, dGTP, dATP, and dCTP ( Pharmacia LKB Biotechnology ), and 1 U Taq polymerase ( Perkin-Elmer Cetus Corp.). The PCR reaction was carried out for 26 cycles of 94°C for 1 min, 60°C for 45 s (or 70°C for 45 s for amplification of JH signal ends), and 72°C for 90 s, followed by a 5-min extension at 72°C. Ligation and PCR amplification with different primers were performed at the same time to minimize experimental variation. Each assay included positive controls and was done several times with independent preparations of DNA. Amplification of PCR products was approximately proportional to the input DNA at several different dilutions. PCR products were separated by electrophoresis and analyzed by Southern blot analysis. Blots were hybridized with: (a) pJH6.3 ( 36 ) to reveal H chain gene rearrangements, DJH coding and signal joints, and unrearranged JH alleles; (b) a genomic fragment corresponding to DHfl and its surrounding region (amplified by PCR using MB222 and MB 241) to reveal LM-PCR–amplified 3′ and 5′ DHfl signal ends; and (c) pActin ( 17 ) to reveal PCR-amplified α actin. Probes were labeled with α-[ 32 P]dCTP using a Prime-It II kit (Stratagene Inc.). Fig. 1 is a schematic representation of the effects of μ and μ/κ tgs on scid B cell development. The different stages of B cell development are designated with the letter code of Hardy et al. ( 17 ); the alternative nomenclature of Rolink and Melchers ( 37 ) is shown for comparison. As indicated, B cell development in scid bone marrow is blocked at stage C, shortly after B lineage cells initiate H chain gene rearrangement. Relief from this block can be achieved by introduction of a μ tg into the scid genome. In μ-tg scid mice ( 22 ), developing pro-B cells appear to bypass stage C and develop directly into early pre-B cells, denoted as C′. Most cells in subset C′ are in cycle ( 17 ) and show downregulated RAG expression ( 23 ). Cells of subset C′ give rise to the D subset. At this late stage of pre-B cell differentiation, RAG expression is again upregulated ( 23 ) and L chain gene rearrangement is initiated ( 17 , 18 , 38 ). Differentiation does not proceed beyond stage D in μ-tg scid mice, presumably because scid pre-B cells are unable to repair DNA DSBs resulting from the initiation of κ gene rearrangement ( 22 , 30 ). Complete relief from the scid block can be achieved in double tg scid mice, bearing both a μ and κ tg ( 20 ). In μ/κ-tg scid bone marrow, B cell development proceeds to stage E and appears to do so very rapidly, as evidenced by the near-normal percentage of B cells and virtual absence of pro- and pre-B cells. Data supporting the above model are illustrated in Figs. 2 and 3 . Fig. 2 shows the effect of two different μ tgs, M54 ( 24 ) and 3H9 ( 25 ), on scid B cell development before the late pre–B cell stage (stage D). Members of subsets C (BP1 + HSA dull ) and C′ (BP1 + HSA bright ), both positive for the early B lineage marker BP1 ( 29 ), are distinguished by their level of staining for heat stable antigen ( 17 ). Note that subset C, which is present in scid mice, appears to be replaced by subset C′ in M54 and 3H9 scid mice. Note also that the BP1 − HSA + cell fraction, which consists exclusively of HSA dull cells in scid mice, includes both HSA dull and HSA bright cells in M54 and 3H9 scid mice. We designate HSA dull and HSA bright cells in the BP1 − HSA + fraction as B and B′, respectively. The upregulation of HSA when μ-tg expressing cells transit from stage B to B′ to C′ presumably reflects μ chain– dependent signaling. The effect of both μ and κ chain tgs on scid B cell development is shown in Fig. 3 . As indicated, scid mice bearing M54 (or 3H9) and the L chain tg, Vκ8 ( 26 ), have near-normal percentages of B (B220 + IgM + ) cells in their bone marrow, but are severely deficient in early B lineage (B220 + CD43 + and B220 + CD43 − ) cells comprising subsets B–D. Since pro-B cells (subsets B and B′) in μ-tg scid mice appear to differentiate directly or very rapidly into early pre-B cells deficient in RAG expression (subset C′), we suspected that many developing B lineage cells in these mice might not initiate DH–JH rearrangement until the late pre–B cell stage (D) when RAG expression is again upregulated. To assay for the initiation of DH–JH rearrangement, we tested for DSBs at JH recombination signal/coding borders in FACS ® -sorted B220 + CD43 + (CD43 + ) and B220 + CD43 − (CD43 − ) bone marrow cells (CD43 + cells would include stages B-C′ and CD43 − cells would correspond to stage D). Broken DNA molecules with JH signal ends were detected by LM-PCR ( 31 , 33 ). We also tested for completed DH-to-JH rearrangements and for retention of JH germline alleles (see Materials and Methods for details). As shown in Fig. 4 and Table I , JH signal ends resulting from the initiation of DH–JH rearrangement in M54 scid mice were much more abundant in the late pre-B (CD43 − ) cell fraction than in the CD43 + fraction containing pro-B and early pre-B cells. In M54 scid/+ mice as well, JH signal ends were more abundant in the CD43 − than CD43 + cell fraction . We conclude that initiation of DH–JH rearrangement in M54 mice occurs predominantly at the late pre–B cell stage. Despite the relatively low abundance of JH signal ends in the CD43 + cell fraction of M54 scid/+ mice, alleles with (completed) DH–JH rearrangements were readily detectable in this cell fraction . This is not surprising as DH–JH rearrangements would be expected to result in DJH complexes that have a much longer half-life than JH signal ends, especially in μ-tg–expressing pro-B cells that fail to rearrange their VH elements ( 20 ). Non-tg scid and scid/+ control mice showed widely different levels of JH signal ends; i.e., JH signal ends were more abundant in the CD43 + cell fraction of scid than scid/+ bone marrow . As discussed later, a possible explanation for this difference is that initiation of DH–JH rearrangement continues unabated in scid mice, whereas in scid/+ mice, DH–JH rearrangement is limited by the onset of VH–DJH rearrangement. JH signal ends were in low abundance in the CD43 − cell fraction of scid/+ mice, consistent with a low retention of germline JH alleles and the completion of H chain gene rearrangement. The high retention of germline JH alleles in the CD43 + cell fraction of scid/+ (and scid) mice is presumed to reflect in part the presence of early B lineage cells not yet expressing RAG protein and the known contamination of this fraction with non–B lineage cells ( 39 ). To test for ongoing DH–JH and VH–DJH rearrangement in the CD43 + and CD43 − cell fractions of scid, scid/+, and M54 scid/+ mice, we assayed for DSBs at both the 3′ and 5′ signals of the DHfl16.1 (DHfl) element. DHfl is the most upstream DH element ( 40 ) and is used in ≥50% of DH–JH rearrangements ( 41 – 44 ). Broken DNA molecules with 3′ DHfl signal ends signify initiation of DH–JH rearrangement, whereas 5′ DHfl signal ends can be taken to reflect initiation of VH–DJH rearrangement ( 33 ). Scid and scid/+ mice showed striking differences in their levels of 3′ and 5′ DHfl signal ends . In the CD43 + cell fraction of scid mice, 3′ but not 5′ DHfl signal ends were abundant, whereas, in the corresponding cell fraction of scid/+ mice, 5′ but not 3′ DHfl ends were abundant. Thus, in the CD43 + cell fraction of scid mice, initiation of DH–JH rearrangement predominates over that of VH–DJH rearrangement, whereas the reverse is true in the CD43 + cell fraction of scid/+ mice. In the late pre-B (CD43 − ) cell fraction of scid/+ mice, neither 3′ nor 5′ DHfl signal ends were detectable, indicating that H chain gene rearrangement is normally completed before this stage, which is in agreement with the results of Fig. 4 . In contrast, in the CD43 − cell fraction of M54 scid/+ mice, DH–JH rearrangement was ongoing, as indicated by the abundance of 3′ DHfl signal ends . Note that 5′ DHfl signal ends were not detectable in the CD43 − (or CD43 + ) cell fraction of M54 scid/+ mice. Therefore, even though DH–JH rearrangement is ongoing in late pre-B cells of M54 scid/+ mice, initiation of VH–DJH rearrangement does not evidently occur in these cells. This apparent inability of the V(D)J recombinase system to target VH elements in late pre-B cells of μ-tg mice is consistent with the early findings of Yancoupolus and Alt ( 45 ). These investigators found that VH558 transcripts are detectable in μ − but not μ + lines of transformed pre-B cells and concluded that VH elements in μ + -transformed pre-B cells are not accessible to the V(D)J recombinase system. To test whether initiation of DH–JH rearrangement occurs at a normal frequency in μ/κ-tg mice, we sorted B220 + IgM − bone marrow cells from scid, 3H9/Vκ8 scid, and 3H9/Vκ8 scid/+ mice, and then assayed for the level of JH signal ends. The B220 + IgM − cell population would include B lineage subsets (B–D) before the immature B cell stage (E). We also assayed for circular DNA molecules with signal joints resulting from the joining of the JH1, JH2, or JH3 signals with the 3′ DHfl signal (see Materials and Methods). Signal joint formation, in contrast to coding joint formation, is not impaired in scid mice ( 46 , 47 ). Also, we would expect circular DNA molecules to have a longer half-life than broken molecules with JH signal ends, thus making signal joint formation a sensitive assay for attempted DH–JH rearrangement in scid mice. As shown in Fig. 6 , JH signal ends were more abundant in the B220 + IgM − cell fraction of non-tg scid mice than in the corresponding cell fraction of 3H9/Vκ8 scid and 3H9/ Vκ8 scid/+ mice. Thus, the initiation of DH–JH rearrangement is clearly reduced in the presence of these tgs. This is also apparent from the reduced level of signal joints in 3H9/Vκ8 mice compared with control non-tg scid mice . The level of JH2 signal joints in 3H9/Vκ8 scid and 3H9/Vκ8 scid/+ mice was estimated to be ∼10 and 60%, respectively, the level in non-tg scid mice . We suggest later that the lower level of signal joints in 3H9/Vκ8 scid than 3H9/Vκ8 scid/+ mice may be attributable to premature death of developing scid B cells resulting from persisting DSBs at DH and JH coding elements. Given that most developing scid B cells fail to rearrange their D and J elements successfully ( 15 , 48 , 49 ) and die with persisting DSBs ( 33 , 35 ), the cells most favored to become B cells in μ/κ-tg scid mice would be those in which DH–JH rearrangement is not attempted. To test this prediction, we generated and cloned B cell hybridomas from the spleen of M54/Vκ8 and 3H9/Vκ8 scid mice, and then examined these hybridomas for the status of their H chain alleles. Control hybridomas were obtained from M54/Vκ8 and 3H9/Vκ8 scid/+ mice. Representative results are illustrated in Fig. 7 for 9 3H9/ Vκ8 scid/+ hybridomas and 10 3H9/Vκ8 scid hybridomas. Note that one or two H chain gene rearrangements were clearly evident in all but one of the scid/+ hybridomas. In contrast, none of the scid hybridomas showed a rearranged allele. 44 hybridomas from μ/κ-tg mice were analyzed and the results are summarized in Table II . 10 scid/+ hybridomas showed one allele to be rearranged with the other allele in germline configuration; 11 (10 of which came from 3H9/ Vκ8 scid/+ mice) showed both alleles to be rearranged and 5 showed a single rearrangement with the other allele missing or undetectable. Two scid/+ hybridomas showed germline H chain alleles only. As normal B cells and their precursors show H chain rearrangements at both alleles ( 3 , 8 , 50 ), the retention of at least one germline H chain allele in ∼40% of the scid/+ hybridomas demonstrates significant transgene-mediated reduction of DH–JH rearrangement. These results are in agreement with the results of Fig. 6 and with previous reports showing that the frequency of endogenous H chain rearrangement is reduced in B lineage cells of M54 ( 10 , 51 ) and 3H9/Vκ8 ( 25 ) wild-type mice. In contrast to the scid/+ results, all M54/Vκ8 and 3H9/ Vκ8 scid hybridomas showed germline H chain alleles only (Table II ). The absence of detectable H chain gene rearrangement in the scid hybridomas indicates that cells that succeed in becoming B cells in μ/κ-tg scid mice do not attempt DH–JH rearrangement. The preceding results show a striking alteration in the representation of B lineage subsets and duration of DH–JH rearrangement in bone marrow of μ-tg and μ/κ-tg scid mice compared with non-tg scid and scid/+ control mice. Specifically, late pro-B cells (subset C) appear to be missing in μ-tg scid mice and DH–JH rearrangement occurs predominantly at stage D, the late pre–B cell stage. In μ/κ-tg scid mice, early B lineage subsets (B–D) are grossly under represented and initiation of DH–JH rearrangement is less frequent than in non-tg control mice. Further, pro-B cells that succeed in reaching the B cell stage in μ/κ-tg scid mice do not attempt DH–JH rearrangement. Interestingly, in non-tg control mice, we found initiation of DH–JH rearrangement to be greater or more sustained in scid mice than in scid/+ mice. The implications of these findings are discussed below. In μ-tg mice initiation of DH–JH rearrangement was found to occur most frequently at the late pre-B cell stage (stage D). As DH–JH rearrangement is normally completed before this stage ( 17 , 18 ), initiation of H chain gene rearrangement appears to be somewhat delayed in μ-tg mice. To explain this result, we suggest the following model: μ-tg- and RAG-expressing pro-B cells (subsets B and B′) rapidly differentiate into recombinase-deficient early pre-B cells (subset C′), such that many cells do not have time to initiate or complete DH–JH rearrangement at both alleles until the late pre-B stage (subset D) when RAG expression is again upregulated. 2 Rapid progression of pro-B cells to the C′ stage would presumably result from premature expression of a pre-BCR containing a tg-encoded μ chain, surrogate light (SL) chain and the signal transducing chains, Igα and Igβ (reviewed in 13 , 52 – 54 ). Consistent with this model is the known early expression of μ tgs ( 30 ) and the genes for SL chain ( 55 – 57 ), the apparent absence of subset C in μ-tg scid mice , the shortened duration of the pro-B stage in μ-tg mice ( 57a ), and the finding that the majority of cells corresponding to subset C in non-tg mice contain nonproductive VDJH (VDJH − ) rearrangements ( 58 ). The latter finding has been interpreted to suggest that pro-B cells containing a VDJH + rearrangement quickly exit the subset C compartment ( 58 ). Applying the above model to non-tg mice, we suggest that pro-B cells that make a VDJH + rearrangement on the first attempt may exclude VH–DJH rearrangement at the other allele by rapidly progressing to the RAG-deficient C′ stage, and then to stage D, at which rearrangement of VH elements is no longer permissible. For allelic exclusion to occur in this model, a pre-BCR need only signal developmental progression. This notion is consistent with previous reports showing that exclusion of VH–DJH rearrangement is tightly linked with progression of pro-B cells to the pre-B stage ( 14 , 59 – 61 ). Such linkage is even observed in μ-tg mice that express a truncated μ chain, which results in a pre-BCR complex lacking (specificity) a μ variable region and surrogate light chain ( 62 , 63 ). However, pro- to pre-B progression and VH–DJH rearrangement are both blocked in μ-tg mice that express a mutated μ chain that precludes assembly of a pre-BCR complex with the signal transducing Ig α/β chains ( 64 – 67 ). Interestingly, the few B lineage cells that reportedly escape the above developmental block show allelic exclusion ( 67 ), consistent with our proposed model. Ongoing initiation of DH–JH and Vκ–Jκ rearrangement ( 30 ) in late pre-B cells of M54 scid mice could help explain why these mice uniformly lack B cells ( 20 ) and appear no more leaky than non-tg scid mice ( 68 ). If attempted rearrangement of DH and JH elements in developing M54 scid pre-B cells is initiated before that of Vκ and Jκ elements, some cells might be expected to succeed in making a DH–JH rearrangement. Indeed, DH–JH rearrangements were recovered from late pre-B cells of M54 scid mice . However, the chance of a scid cell surviving attempted rearrangements at both H and L chain loci would seem unlikely, consistent with the absence of detectable VJκ coding joints in late pre-B cells of M54 scid mice ( 22 , 30 ). In μ/κ-tg mice, we found initiation of DH–JH rearrangement was less frequent than in non-tg scid mice. Signal joints resulting from the initiation of DHfl to JH rearrangement in 3H9/Vκ8 scid and 3H9/Vκ8 scid/+ mice were estimated to be present at ∼10 and 60%, respectively, the level observed in non-tg scid mice. Based on the difference in level of recovered signal joints in 3H9/Vκ8 scid/+ and non-tg scid mice, we estimate that initiation of DH–JH is ∼40% less frequent in μ/κ-tg than non-tg mice. This estimate agrees favorably with the observed frequency of germline H chain alleles in B cell hybridomas from 3H9/Vκ8 (6/35 alleles) and M54/Vκ8 (6/14 alleles) scid/+ mice (Table II ). The much lower level of signal joints in 3H9/Vκ8 scid than 3H9/Vκ8 scid/+ mice is taken to reflect loss (death) of scid cells that attempt DH–JH rearrangement. This could account for the absence of rearranged H chain alleles in B cell hybridomas recovered from μ/κ-tg scid mice (Table II ). To explain the reduced level of DH–JH rearrangement in μ/κ-tg mice, we suggest that expression of a tg-coded BCR in early pro-B cells promotes very rapid progression of these cells to the B cell stage, such that there is little time to initiate DH–JH rearrangement. Consistent with this notion, (a) μ and Vκ8 tgs are known to be expressed early in B cell development ( 30 ), (b) μ/κ-tg scid/+ mice contain near-normal percentages of B cells in bone marrow but markedly reduced percentages of pro- and pre-B cells ( 20 ), and (c) μ/κ-tg scid mice show near-normal percentages of B cells in bone marrow but sharply reduced percentages of pro-B cells compared with non-tg scid controls and virtually no pre-B cells . In non-tg control mice, we found that early B lineage cells in the CD43 + cell fraction from scid mice showed a much higher level of JH signal ends than the corresponding cell fraction from scid/+ mice. As scid does not impair the joining of signal ends ( 46 , 47 ), one cannot attribute the relatively high level of JH signal ends in scid mice to a blockage in signal joint formation. What scid does impair, however, is the processing of coding ends before their being joined ( 33 , 35 ). Thus, developing B lineage cells in scid mice do not often succeed in joining DH and JH coding ends ( 48 , 49 ) and would not be expected to initiate the second step of H chain gene rearrangement (VH–DJH rearrangement). Indeed, 5′ DHfl signal ends, signifying the initiation of VH–DJH rearrangement, were not detectable in CD43 + scid cells . We suggest that in the absence of VH–DJH rearrangement, initiation of DH–JH rearrangement continues unabated in CD43 + cells, resulting in a high level of JH signal ends. On the other hand, in the CD43 + cell fraction of scid/+ mice, initiation of VH–DJH rearrangement was prominent and that of DH–JH rearrangement barely evident . This implies that initiation of DH–JH rearrangement in scid/+ mice may be limited to the earliest stage of pro-B cell development, consistent with the idea discussed below, that onset of VH–DJH rearrangement may preclude further DH–JH rearrangement. In wild-type or scid/+ cells, a DH–JH rearrangement may be followed by rearrangement of a VH element to the resulting DJH complex or the complex may be replaced by the joining of an upstream DH element to a downstream JH element ( 41 ). The latter event, DJH replacement would seem counterproductive to efficient assembly of VH, DH, and JH elements. Thus, it makes sense, as originally postulated by Alt et al. ( 69 ), that after DH–JH rearrangement VH rather than DH elements are preferentially rearranged. How might this happen? Recent evidence suggests that initiation of VH–DJH rearrangement is associated with a shift in the targeting of the V(D)J recombinase activity from the 3′ to the 5′ side of DH elements ( 70 ). Targeting of the recombinase to signals on the 5′ side of DH elements would minimize DJH replacement and limit the duration of DH–JH rearrangement to the earliest stage of pro-B cell development. Although DJH complexes can be readily detected in late pro-B cells (subset C) ( 17 , 18 , 38 ), this does not necessarily reflect ongoing DH–JH rearrangement at this stage; the observed DJH complexes could have been formed earlier in cells of subset B. In sum, DH–JH rearrangement in non-tg mice is normally initiated and completed at the early pro-B stage. In μ-tg mice, DH–JH rearrangement may begin at the pro-B stage, but it appears to continue and occur most frequently at the late pre-B stage. Based on the altered representation of pro-B subsets in μ-tg scid mice, we suggest that the extended period of DH–JH rearrangement in these mice primarily reflects rapid progression of μ-tg–expressing pro-B cells to the recombinase-deficient early pre-B cell stage. Thus, many cells may not have time to initiate DH–JH rearrangement until the late pre-B stage when RAG expression is again upregulated. In addition, ongoing DH–JH rearrangement (including DJH replacement) at the late pre-B stage would not be limited by initiation of VH–DJH rearrangement, as the latter does not apparently occur in late pre-B cells of μ-tg mice. Finally, rapid progression of μ/κ-tg–expressing pro-B cells to the recombinase-inactive B cell stage could explain why in μ/κ-tg mice we find a reduced initiation of DH–JH rearrangement compared with non-tg mice and a striking deficiency of pro- and pre-B cells despite near-normal numbers of B cells. | Study | biomedical | en | 0.999998 |
10209047 | Mice were generated from chimeras established using a previously described embryonic stem cell (ES) clone ( 11 ) containing a targeted integration of a tk-neo cassette into Cd22 . The chimeras (created using C57BL/6 blastocysts) were bred against both C57BL/6 and BALB/c mice, and mice from the F2 generation were maintained for up to 20 mo with tail bleeds taken every 4–6 wk. A cohort of control (129 × C57BL/6)F2 mice (that do not carry any targeted gene alteration) was established analogously. Animals were either bred in our own conventional facility or in a specific pathogen-free (barrier) unit following delivery by Caesarian section and fostering onto C57BL/6 × CBA females in isolators. Serum titers of IgG anti–double-stranded (ds) DNA were measured as described elsewhere ( 20 ) using alkaline phosphatase–conjugated goat anti–mouse IgG ( Sigma Chemical Co. , Ltd.). Sera from four MRL/lpr mice were always titered in parallel, with one of these sera assigned a titer of 5 U/ml. The assay was calibrated using a high affinity IgG2a monoclonal anti-dsDNA antibody (S22) from mouse 9612 (see below); 1 U/ml in the ELISA was given by 24 μg/ml of S22. Titers of other IgG autoantibodies were similarly determined using plates that had been coated with either cardiolipin ( Sigma Chemical Co. ; 100 μg/ml in ethanol) or myeloperoxidase ( Calbiochem Corp. ; 250 ng/ml in sodium bicarbonate, pH 9.2). Antibody isotypes were determined using reagents from PharMingen . Hybridomas were established from unimmunized mice by fusion with NS0 cells and autoantibodies in the supernatants monitored by ELISA developing with biotinylated goat anti–mouse κ (Southern Biotech) and alkaline phosphatase–conjugated streptavidin (Dako). The binding of monoclonal anti-DNA antibodies at 20°C to a 5′-biotinylated ds 48mer oligonucleotide that had been immobilized on a streptavidin-coated chip (SA-Biacore chip; Pharmacia ) was monitored by surface plasmon resonance as previously described ( 21 ). Oligo-dT–primed cDNA prepared from RNA extracted from the hybridomas was PCR amplified using a consensus V H oligonucleotide for forward priming (5′-CGGGATCCTGAGGTGCAGCTGGAGGAGTC ) in conjunction with either 5′-CGGAATTCGGGGCCAGTGGATAGAC or 5′-CGGAATTCGGGACCAAGGGATAGAC for priming back from the C H 1 domain of Cγ1, γ2a, and γb or of Cγ3, respectively. PCR products were sequenced directly as well as ligated into pUC18 with multiple DNA clones sequenced from each hybridoma. We have previously described ( 11 ) an ES line (derived from 129 mice) that carries a targeted integration of a neomycin resistance gene into the CD22 gene; this cell line was used to establish chimeric mice by injection into C57BL/6 blastocysts and germline transmission of the targeted allele (yielding CD22 +/− heterozygotes) obtained following breeding with both C57BL/6 and BALB/c females. Cohorts of animals from the F2 generations of both sets of breedings were followed with time for the development of IgG anti-dsDNA antibody. On both backgrounds, high titers of anti-DNA antibodies developed with age (particularly after 8 mo) in many of the CD22 −/− animals but not in the CD22 +/+ litter-matched controls. That the development of these autoantibodies was due to the targeted integration into the CD22 gene is confirmed by the fact that IgG anti-dsDNA was not detected in the sera of control (129 × C57BL/6)F2 mice . The titer of anti-DNA antibody in the CD22-deficient mice is, in many cases, of a comparable order to that found in 12-mo-old MRL/lpr mice. By 18 mo of age, over 70% of the CD22-deficient mice have at some time shown evidence of IgG anti-dsDNA antibody in their sera at concentrations >1.5 U/ml; none of the 42 control mice revealed titers of this magnitude . We have also followed a limited number of CD22 +/− heterozygotes and found that 3/11 had developed IgG anti-dsDNA by 12 mo of age (not shown). Life expectancy among CD22-deficient mice was decreased (10/43 weaned CD22-deficient mice having died by 15 mo of age compared with 1/43 CD22 +/+ controls), with at least 4 of the deaths due to infection. However, all but one of these deaths occurred after 7 mo of age. Furthermore, we did not detect proteinuria or antibody deposition in glomeruli in the mice harboring autoantibodies. This lack of pathology may well correlate with the fact that anti-DNA titers do not simply rise with age but, in individual animals, often rise, regress, and rise again. dsDNA is not the sole target of autoantibody development. Mice were also monitored for the development of antibodies to cardiolipin and myeloperoxidase; a clear distinction between the CD22-deficient and control siblings was found here as well . The largest cohort of animals was followed under conditions of conventional housing, but we also compared autoantibody development in CD22-deficient and control mice housed in a barrier unit. The results (Table I ) reveal that autoantibodies also develop under these cleaner conditions. Subclass typing of serum autoantibodies revealed that, in both the C57BL/6 and BALB/c breedings, IgG2a anti-DNA was found in ∼80% of the autoimmune animals. However, >50% of the autoimmune mice contained anti-DNA antibodies of multiple IgG subclasses. To obtain more detail about the nature of these antibodies, hybridomas were established from two unimmunized, 18-mo-old, CD22-deficient females , as well as two CD22 +/+ litter-matched controls. No anti-dsDNA IgG was detected in the supernatants from 198 wells from the control fusions; strong titers, however, were detected in 20/302 wells from the CD22-deficient mice . Similarly, whereas no cardiolipin-specific hybrids were detected in the control fusions, 19 positives were obtained from the CD22-deficient mice . The majority of these cardiolipin-specific antibodies were IgMs, although mouse 9612 gave two IgA and mouse 9449 gave two IgG3 anticardiolipin antibodies. The hybridomas were then expanded for further characterization. Analysis of the anti-DNA antibodies by surface plasmon resonance using a biotinylated oligonucleotide as antigen revealed that several bound DNA very tightly, with dissociation half-lives in the range of 8–500 min . To ascertain whether the B cells producing these antibodies were clonally related and whether they had undergone somatic hypermutation, the V H sequences were determined from several of the anti-DNA antibodies from mouse 9612. The results demonstrate that the anti-DNA antibodies within a single CD22-deficient mouse derive from multiple, clonally expanded B cell progenitors that have undergone class switching and somatic hypermutation. Thus, for example, hybridoma S48 appears to be derived from S30, as they carry the same V H 36–60/J H 2 rearrangement but with S48 harboring multiple additional somatic mutations (several to arginine), which could account for its increased affinity . Similarly, S31 (IgG1) and S35, S66, S11, and S15 (all IgG2a) all express the same (V H J558 family member)/J H 2 rearrangement with an arginine-rich CDR3 (characteristic of many anti-DNA antibodies [23–25]); the individual antibodies differ, however, in the extent of accumulated somatic mutations . In contrast, S20 and S22 carry distinct rearrangements of the same V H 7183 family member . Thus, paralleling observations previously made with other autoimmune mice ( 25 ), multiple independent B cells appear to have seeded an ongoing anti-DNA response. Similarly, in respect of the two cardiolipin-specific IgG3s, analysis of their V H sequences revealed them to be clonally related . The development of autoantibodies in CD22-deficient mice reveals that a single gene defect exclusive to B cells is sufficient to trigger autoimmunity in a large proportion of mice. This presumably means that the restriction of the provision of T cell help to foreign antigens is intrinsically imperfect: T cell help for an autoantibody response can be elicited by a hyperreactive B cell compartment. The CD22-deficient animals do not, however, go on to develop autoimmune disease. This is consistent with genetic analyses of predisposition to systemic autoimmune disease in lupus-prone mouse strains, which reveal a role for multiple genetic loci ( 1 – 4 , 26 ). Indeed, one of the loci contributing to autoimmunity in NZM mice ( Sle3 ) has been mapped to a region of chromosome 7 in the vicinity of Cd22 and, when bred into C57BL/6 mice, causes production of IgG anti-dsDNA antibodies as well as lupus nephritis ( 27 ). It will be interesting to ascertain whether this, at least in part, reflects a functionally relevant Cd22 polymorphism. By analogy with studies in the MRL mouse ( 28 ), it will also be interesting to ascertain whether mutations in Fas or its ligand exacerbate autoimmunity in CD22-deficient mice. The precise mechanism by which CD22 deficiency predisposes to autoimmunity remains to be definitively identified, but we believe the hyperresponsiveness of CD22-deficient B cells to BCR ligation is likely to be of central importance. Phosphorylation of CD22 on its cytoplasmic tyrosines following BCR ligation is mediated by the Lyn kinase and leads to the recruitment of the phosphatase SHP1 ( 29 – 34 ). It is therefore notable that deficiencies in either Lyn or SHP1 both lead to autoimmunity ( 35 – 38 ). However, this autoimmunity is more severe than that in CD22-deficient animals and is most unlikely to simply reflect defects in CD22-mediated regulation of BCR. Indeed, the increased severity probably correlates with both Lyn and SHP-1 being implicated in signal transduction through multiple cell-surface receptors, with their functions not being limited to the B cell lineage. Thus, the significance of the autoantibody development in CD22-deficient mice lies in the fact that these autoantibodies arise as a consequence of a relatively mild perturbation that is exclusive to B lymphocytes and that affects the BCR signaling threshold. Experiments performed using transgenic mice that have been engineered to express high affinity autoreactive specificities on a substantial proportion of their B cells have revealed that the fate of such B cells is sensitive to modifications in CD22, Lyn, and SHP-1 as well as other genes that affect BCR signaling ( 9 , 10 ). Our findings are entirely consistent with these earlier studies but reveal that CD22 deficiency alone, without additional contrivance, is sufficient to predispose autoimmunity in normal animals. It is attractive to speculate from our results that the major physiological function served by CD22 in normal mice is to mediate the avoidance of autoimmunity. In light of the diminished level of CD22 expression in immature B cells ( 39 ), we previously suggested ( 11 ) that CD22 plays a role in raising the threshold of sensitivity to antigen that accompanies differentiation of an immature B cell (sensitive to tolerization/deletion by low affinity antigen) into a mature B cell that awaits triggering by exogenous antigen ( 40 ). Such a proposal could well explain the autoimmunity in CD22-deficient mice. However, a role for CD22 should also take into account the specificity of its extracellular domain for α-2,6-sialoglycoconjugates ( 18 ). Intriguingly, the sialylated moieties present on eukaryotic membranes enhance the interaction between complement components C3b and factor H, thereby leading to inhibition of the alternative complement pathway; this serves to bias activation of the innate immune system toward microbial infection and away from autoreactivity ( 41 , 42 ). Maybe CD22 recognition of the sialoglycoconjugates expressed on mammalian cells serves an analogous role in the adaptive immune system, dampening the BCR signaling that might otherwise be triggered by low affinity autoantigens. It will be interesting to ascertain whether making mutations in the CD22 extracellular domain that abolish recognition of sialoglycoconjugates will be sufficient to predispose autoimmunity. | Study | biomedical | en | 0.999997 |
10209048 | Mice deficient in IL-12, as a result of targeted disruption of the IL-12p35 gene (IL-12p35 KO), were generated and bred at Genetics Institute, using the p35 SK+ vector (Stratagene Inc.), the embryonic stem cell system in 129/sv mice, and established procedures ( 30 – 32 ). Mice were backcrossed onto the C57BL/6 genetic background for two to four generations. Homozygous mutant (−/− or KO) and homozygous wild-type (WT) IL-12p35 gene mice were taken from littermates for use in these studies. The gene targeting vector eliminated exons 1–4 by replacement with the neomycin resistance gene . Disruption of the p35 gene was demonstrated by Southern blot analysis . Functional deletion of IL-12 was confirmed by absence of IL-12p70 heterodimers after challenge with the known stimulators lipopolysaccharide treatment and MCMV infection. For these studies, mice were injected at 0 h with 500 μg/kg LPS ( 3 ), and blood collected for serum sample preparation at 2.5 and 5 h , or infected with 5 × 10 4 PFU of Smith strain MCMV V70 salivary gland extract, and blood collected for serum sample preparation at 36 h . Lack of IL-12 was shown to have biological consequences because the IL-12–dependent induction of natural killer cell IFN-γ production after MCMV infection was blocked in the IL-12p35 KO compared with WT mice . Mice deficient in IFN-α/β receptor function as a result of genetic mutation (IFN-α/βR KO) on the 129/Sv background ( 33 , 34 ), originally obtained from B&K Universal Limited, were bred and maintained in strict isolation in the animal facility at Brown University. Age-matched WT 129 control (129 SvEvTacFBR) mice were purchased from Taconic Farms Inc. All mice used in experiments had the H-2 b major histocompatibility complex and were handled in accordance with institutional guidelines for animal care and use. The LCMV infections were established i.p. on day 0 with 2 × 10 4 PFU of Armstrong strain, clone E350 ( 11 , 13 , 35 , 36 ). CD8 T cells were depleted in vivo by treatment i.p. with 0.5 mg of monoclonal anti–CD8 antibody 2.43, prepared from ascites, on day 5 after infection. Control treatment was with partially purified P3NS1 ascites containing 0.5 mg rat IgG ( Sigma Chemical Co. ). Sheep antimurine IFN-α/β IgG was injected (0.15 mg/mouse) to neutralize IFN-α/β with nonspecific sheep IgG used as control antibody (gifts of Ion Gresser, Center National de la Recherche Scientifique, Villejuif, France) ( 10 , 13 , 37 , 38 ). The rat IgG monoclonal directed against the mouse IL-12 p40 chain, C17.8, was used to neutralize IL-12 function in vivo. Partially purified ascites preparations (1 mg) were injected to neutralize IL-12 with rat IgG added to P3NS1 ascites used as control antibody ( 10 , 11 ). Injections were i.p. 14 h before, and day 4 of, LCMV infections. Splenic leukocytes were isolated from macerated whole spleens after passage through nylon mesh and osmotic lysis of erythrocytes by ammonium chloride treatment, with viable cell yields determined by trypan blue exclusion. Samples for quantitation of IFN-γ and IL-12 p70 were prepared as reported previously ( 11 , 13 , 17 ). Conditioned media (CM) were generated by incubating 10 7 splenic leukocytes/ml in 10% FBS-RPMI 1640 media for 24 h at 37°C before harvest of culture supernatants. Where indicated, CM samples were prepared by incubation of splenic leukocytes with or without 0.1 μg/ml LCMV peptides NP396-404 or GP33-41 in 48-well flat-bottom plates at concentrations of 2 × 10 6 cells per well in 0.4 ml of 10% FBS-RPMI supplemented with 50 U/ml human recombinant IL-2 ( 23 , 25 , 39 ). IFN-γ was measured by sandwich ELISA ( 11 , 13 , 17 ). Standard curves of mrIFN-γ ( PharMingen ) indicated detection limits of 10–40 pg/ml in serum, or 1–4 pg/10 6 cells in CM. IL-12 p70 was quantitated either in an ELISA ( Genzyme Corp. ) or in a biological assay ( 11 , 13 ). Limit of detection for IL-12 p70 in the biological assay was 0.7 pg/ml serum. IL-2 was quantitated in ELISA using reagents from PharMingen according to the manufacturer's recommendations. As per modification of published techniques ( 40 ), cells were stimulated on 24-well cluster plates previously coated overnight with 0.5 ml 10 μg/ml purified hamster anti–mouse CD3ε mAb 145-2C11 ( PharMingen ) in PBS. Plates were washed with PBS, and 2 × 10 6 cells in 2 ml 10% FBS-RPMI per well were incubated for a total of 6 h, with Brefeldin A ( Sigma Chemical Co. ) added the last 2 h. Alternatively, splenic leukocytes were stimulated with 0.1 μg/ml of the LCMV peptides NP396-404 or GP33-41 ( 23 ). Cells for these experiments were incubated in 48-well flat-bottom plates at concentrations of 2 × 10 6 cells per well in 0.4 ml of 10% FBS-RPMI supplemented with 50 U/ml human recombinant IL-2, at 37°C for 5 h total, with Brefeldin A added for the last 3 h. After stimulation, cells were washed in staining buffer (0.5% BSA and 0.006% NaN 3 in PBS), incubated for 30 min at 4°C with biotinylated rat anti–CD4 mAb clone RM4-5 ( PharMingen ), washed twice with staining buffer, incubated 30 min at 4°C with streptavidin-PerCP ( Becton Dickinson & Co.) and anti–CD8α FITC-conjugated rat mAb 53-6.7 ( PharMingen ), washed once with staining buffer, once with PBS, resuspended at 10 6 cells/ml, and fixed with an equal volume of 4% formaldehyde in PBS for 20 min at room temperature. For cytoplasmic staining, cells were washed once with PBS, once with staining buffer, treated with 150 μl of permeabilization buffer (1% saponin in staining buffer; Sigma Chemical Co. ), resuspended in 25 μl permeabilization buffer containing 300 μg/ml rat IgG ( Sigma Chemical Co. ), incubated 10 min at room temperature, and incubated for 20 min at room temperature with anti–IFN-γ PE-conjugated rat mAb XMG1.2 ( PharMingen ). Specificity of IFN-γ staining was established by preincubation of anti–IFN-γ-PE with recombinant murine IFN-γ at 0.25 mg/test, before incubation with cells (cold block), or preincubation of cells with excess unconjugated XMG1.2 (cold competitor), and nonspecific staining assessed by use of control antibodies lacking specificities for murine determinants ( PharMingen ). Cells were washed twice with permeabilization buffer, once with staining buffer, and acquired immediately. H2D b tetramers, containing LCMV peptides NP396-404, GP33-41, and GP276-286, prepared, and conjugated with allophycocyanin as described, were used for surface staining along with anti–CD8 PE-conjugated rat mAb 52-6.7 ( PharMingen ) ( 23 , 41 ). After incubation of 10 6 cells with tetramer for 1 h, cells were washed two times in 1× PBS supplemented with 2% FBS, fixed with 4% paraformaldehyde and washed one time. All samples were acquired at Brown University on a FACSCalibur ® ( Becton Dickinson & Co.), using CELLQUEST 3.0 software. More than 20,000 events were collected, with laser outputs of 15 mW at 488 or 635 nM. Livers and spleens were frozen at −80°C, thawed, homogenized, and LCMV titers were measured by plaque formation on Vero cells as previously described ( 17 , 35 , 36 ). To conclusively exclude a role for IL-12 in T cell responses to LCMV infection, mice were made deficient for the p35 subunit of IL-12 (IL-12p35 KO) by homologous recombination with a deleted gene construct, as described in Materials and Methods and Fig. 1 . None of the day 8 splenic T cell responses to LCMV infection were significantly reduced in the IL-12p35 KO mice. Yield and flow cytometric analyses demonstrated that overall expansion of CD8 T cells was similar in both WT and IL-12p35 KO . Furthermore, ELISA studies demonstrated that induction of IFN-γ expression, both in media conditioned with splenic leukocytes (CM) and in serum, was neither blocked nor significantly inhibited in the absence of IL-12 . Because CD8 T cells are the predominant IFN-γ producers during LCMV infection, IFN-γ expression by CD8 T cells also was measured by flow cytometric analysis for cytoplasmic protein and shown to be unaffected by absence of IL-12. Upon stimulation ex vivo with immobilized anti–CD3, ∼60–70% of the cells induced to express cytoplasmic IFN-γ from both types of mice were CD8 T cells, and the proportions of IFN-γ–expressing CD8 T cells were 43.2% (± 8.0) and 43.5% (± 4.7) (means ± SEM) in WT and IL-12p35 KO mice, respectively . Likewise, total numbers of CD8 T cells expressing IFN-γ, calculated based on CD8 T cell yields, were equivalent with 7.9 (± 2.2) and 9.6 (± 2.7) × 10 6 cells . The endogenous responses were sufficient to mediate protection against infection because viral titers were below detectable levels in both infected WT and IL-12p35 KO mice by day 8 (data not shown). Thus, T cell proliferation and IFN-γ production occur in the complete absence of the biologically active IL-12 heterodimer during infections with this virus, and the immune responses induced under these conditions are protective. Experiments were carried out to characterize roles of IFN-α/β in regulating the T cell responses. Although of lower magnitude on the inbred genetic 129 background of these mice, inductions of CD8 T cell expansion and IFN-γ expression were observed and similar in both WT and IFN-α/βR KO mice . IFN-γ levels, measured in samples from the IFN-α/βR KO mice, were equal to or greater than those from WT mice in CM and serum . Interestingly, serum IFN-γ levels were enhanced significantly by more than threefold in samples obtained from IFN-α/βR KO, as compared with WT, mice. Studies, in IFN-α/βR KO mice depleted of endogenous CD8 T cells by antibody treatments, demonstrated that 66% of the enhanced serum IFN-γ was dependent on endogenous CD8 T cells. After anti–CD3 stimulation ex vivo, 50–60% of the IFN-γ expressing cell types from both IFN-α/βR KO and WT mice were CD8 T cells (data not shown), and similar proportions of the CD8 T cell subset from both types of mice were induced to express the cytokine . As these T cell responses are intact, the data suggest that IFN-α/β deficiencies by themselves do not result in generalized T cell exhaustion or depletion. However, viral titers were increased in IFN-α/βR KO relative to WT mice (see below). Hence, the CD8 T cell expansion and IFN-γ production are not IFN-α/β dependent, but the conditions result in decreased resistance to infection. Because total expansion and IFN-γ expression were intact, but levels of virus were increased, antigen specificity of the responding cells was tested. To enumerate CD8 cells bearing T cell receptors for LCMV antigens, splenic leukocytes were labeled with anti–CD8 and tetramers of D b MHC class I molecules containing LCMV peptides immunodominant for CD8 T cell responses (NP396-404 or GP33-41), as described in Materials and Methods. Flow cytometric analyses showed significant increases in percentages of CD8 T cells binding tetrameric D b NP396-404 or D b GP33-41 with cells from day 8 LCMV-infected, relative to uninfected, WT mice . Samples from IFN-α/βR KO mice also had significant increases in proportions of tetrameric D b NP396-404– and D b GP33-41–binding CD8 T cells after infection . T cell frequencies specific for tetramers with NP396-404 were higher in WT, whereas those with GP33-41 were higher in IFN-α/βR KO, populations. Total numbers of CD8 T cells binding the complexed tetramers were significantly increased after infection, relative to uninfected samples, in both WT and IFN-α/βR KO mice . Moreover, because of increases in average splenic cell yields after infection, the total numbers of CD8 T cells specific for the tetrameric D b LCMV peptide complexes were higher in the IFN-α/βR KO than the WT mice, respectively, averaging 247 and 120 × 10 4 cells per spleen. As a result of their skewed proportional representation, however, the D b GP33-41 binding cells respectively comprised ∼75 and 20% of the populations, and appeared to account for a large proportion of the enhanced expansion of T cells recognizing viral epitopes in the IFN-α/βR KO mice. Thus, as in WT mice, expanding CD8 T cells in IFN-α/βR KO mice are specific for the virus. However, in the absence of IFN-α/β function, responses are differentially elicited to particular LCMV epitopes. To demonstrate specificity of functional CD8 T cells, IFN-γ expression responses to the LCMV NP396-404 and GP33-41 peptides were evaluated ex vivo. Cells from uninfected WT or IFN-α/βR KO mice were not stimulated by either of these peptides (data not shown). In contrast, on day 8 after LCMV infection, significant increases in CD8 T cells expressing cytoplasmic IFN-γ, relative to samples prepared in the absence of exogenous stimulation, were observed after peptide stimulation of either WT or IFN-α/βR KO splenic leukocytes . Consistent with the Fig. 2 H experiments, proportions of total primed CD8 T cells responding to anti–CD3 stimulation for IFN-γ expression remained similar . However, even though the total proportions of cells specific for binding tetramers with LCMV peptides , and of cells expressing cytoplasmic IFN-γ after anti–CD3 stimulation , were similar, lower proportions of cells from IFN-α/βR KO as compared with WT mice were stimulated in response to peptides . Interestingly, despite dramatic increases in cells binding tetrameric D b GP33-41 , the proportions and numbers of cells responding to this peptide for IFN-γ expression were similar or only marginally higher in IFN-α/βR KO compared with WT populations . Thus, the reduced IFN-γ responses with cells from IFN-α/βR KO mice appeared to be a consequence of specifically binding populations failing to detectably respond to GP33-41 stimulation for IFN-γ expression. Evaluation of cytokine in CM demonstrated similar levels of population responsiveness to the LCMV peptides (Table I ). These studies demonstrate induction of LCMV-specific CD8 T cell IFN-γ production responses in the absence of IFN-α/β functions. Taken together with the tetramer binding results, they provide evidence that a proportion of the CD8 T cells specifically expanding in response to viral epitopes are altered in their requirements for stimulation and/or magnitude of functional responses. Kinetic studies of viral clearance during the experiments reported here, sampling on various days after LCMV infection, demonstrated that viral burdens in both IL-12p35 KO (data not shown) and WT mice were below detection by day 8 after infection. Extended studies of WT and IFN-α/βR KO mice showed that WT mice had viral burdens that peaked around day 4.5 at levels of 6.2 (± 0.2) and 5.0 (± 0.2) log PFU/g of spleen and liver, respectively, and were below detection by day 7 . Similar kinetics of splenic and hepatic viral titers were observed in IFN-α/βR KO mice, but these peaked at the higher levels of 10.3 (± 0.2) and 10.5 (± 0.0) log PFU/g of respective tissue on day 4.5 after infection, and declined by day 7 . Interestingly, decreases in splenic and liver viral loads were observed in the IFN-α/βR KO mice over time to 4.7 (± 0.2) and 4.2 (± 0.0) log PFU/g of tissue, respectively, on day 14 after infection, and below the limits of detection by day 28 after infection . Thus, absence of an endogenous IL-12 response does not increase sensitivity to LCMV infection, and although absence of IFN-α/β functions does, viral clearance is eventually achieved. Because IL-12 expression can be revealed during LCMV infection by neutralization of endogenous IFN-α/β functions ( 13 ), a released IL-12 induction may substitute in supporting T cell responses under these conditions. Measurements of IL-12 p70 in serum demonstrated that the factor was induced to detectable levels on days 1.5 and 3 of LCMV infection in IFN-α/βR KO, but not WT, mice . Therefore, IL-12 effects on the T cell responses were examined in IFN-α/βR KO mice by treatments with antibodies neutralizing IL-12. Although anti–IL-12 treatments did not modify IFN-γ production responses as compared with control antibody treatments in WT mice, they significantly reduced IFN-γ production on day 8 in LCMV- infected IFN-α/βR KO mice; relative to cells isolated from control-treated IFN-α/βR KO mice, 70% decreases in CM spontaneous production of IFN-γ were observed . Furthermore, anti–IL-12 treatment of IFN-α/βR KO mice, resulted in a >85% reduction in CM IFN-γ levels, as compared with anti–IL-12–treated WT mice ; i.e., from 66.4 (± 16.0) to 7.8 (± 2.0) pg/10 6 cells. Consistent with the experiments shown in Fig. 2 , B and E, only low levels of serum IFN-γ were detected in WT mice, but were increased by more than threefold in IFN-α/βR KO mice . Given the contribution of CD8 T cells to serum IFN-γ (see above), the stimulation of CD8 T cell IFN-γ expression by LCMV epitopes , and the significant increases in viral burdens , elevated in vivo stimulation of the CD8 T cells by LCMV epitopes was likely to have contributed to higher serum IFN-γ levels in the IFN-α/βR KO mice. IL-12 also participated in this enhanced response because neutralization of the factor resulted in a 40% reduction in serum IFN-γ levels . Thus, in the absence of endogenous IFN-α/β effects during a viral infection, IL-12 can be induced and substitute to provide conditions supporting IFN-γ responses. To confirm and extend these studies, IFN-α/β effects were examined under the reciprocal conditions in mice lacking endogenous IL-12. For these experiments, WT and IL-12p35 KO mice were treated with antibodies neutralizing IFN-α/β or control antibodies. Cytokine neutralization of WT mice resulted in IFN-γ responses comparable with those in IFN-α/βR KO mice; i.e., IFN-γ production in CM was not blocked and was induced to elevated levels in serum . Relative to control-treated mice, antibody-mediated neutralization of IFN-α/β in IL-12p35 KO mice significantly inhibited IFN-γ levels spontaneously produced in CM . Moreover, in comparison with anti– IFN-α/β–treated WT mice, serum IFN-γ levels were reduced by >90% as a result of IFN-α/β neutralization in the IL-12p35 KO mice; i.e., whereas the anti–IFN-α/β– treated WT mice had levels of 1,454.2 (± 171.2) pg/ml in serum, the anti–IFN-α/β–treated IL-12p35 KO mice had only 110.9 (± 30.6) . Thus, in the absence of endogenous IL-12, IFN-α/β–mediated effects are primarily responsible for endogenous conditions promoting the IFN-γ responses to viral infections. To evaluate the specificity of CD8 T cell responses for LCMV, under the conditions of anti–IL-12 treatment in IFN-α/βR KO mice, experiments were carried out examining peptide or anti–CD3 stimulation for cytoplasmic IFN-γ expression, specific stimulation by peptides for IFN-γ production in CM, and surface binding of tetramers complexed with peptides immunodominant for CD8 T cells. Overall, both the control and anti–IL-12 antibody treatments modestly blunted magnitudes of the ex vivo– detected specific responses. As a result, intracytoplasmic labeling was not sensitive enough to identify changes in the low proportions of CD8 T cells specifically responding to peptides with cytoplasmic IFN-γ expression . However, it was possible to demonstrate decreases, resulting from the blocking IL-12 function in IFN-α/βR KO mice, in the proportion of CD8 T cells primed for anti– CD3 stimulation of IFN-γ expression; i.e., 18.3% (± 2.2) to 12.8% (± 0.2). The anti–IL-12 treatments also resulted in significant 46 and 53% inhibitions of NP396-404– and GP33-41–stimulated IFN-γ production, respectively, in CM . In contrast to the effects on IFN-γ responses, blocking of both cytokine pathways did not inhibit LCMV-induced CD8 T cell expansion; increased proportions and numbers of CD8 T cells specific for D b NP396-404 and D b GP33-41 were observed with or without IL-12 neutralization (data not shown). These studies demonstrate that the conditions of a revealed IL-12 pathway, in the absence of IFN-α/β–mediated functions, result in IFN-γ production promoted in a virus-specific manner by epitopes immunodominant for CD8 T cell responses. In contrast, the results indicate that specific CD8 T cell proliferation can occur in the absence of both IL-12 and IFN-α/β–mediated effects. To assess the contribution of IL-12 to virus clearance in the absence of IFN-α/β function, viral titers were measured in LCMV-infected mice having had both factors blocked. Anti–IL-12 treatment of IFN-α/βR KO mice resulted in statistically significant ( P < 0.01) increases in viral titers of almost 1 log by day 14 after infection; spleens and livers from anti–IL-12–treated IFN-α/βR KO had 5.8 (± 0.0) and 5.3 (± 0.1) log PFU/g, respectively, as compared with the 5.1 (± 0.1) and 4.4 (± 0.1) log PFU/g of tissue observed in control-treated IFN-α/βR KO mice (means of three mice per group ± SEM). These results show that the endogenous IL-12 response in IFN-α/βR KO mice promotes the antiviral state of the host, but cannot substitute for endogenous IFN-α/β in clearing virus expediently. These studies have characterized divergent innate pathways for promoting IFN-γ responses during viral infections, with induction of high level IFN-α/β resulting in conditions supporting one and acting to limit a potential alternative IL-12 pathway. The experiments demonstrate (a) an IFN-α/β pathway for IFN-γ induction, and (b) IL-12 independence of the CD8 T cell responses of expansion and IFN-γ production, during LCMV infections. Moreover, they show that in the absence of endogenous IFN-α/β– mediated functions, an IL-12 response is revealed and sufficient to support induction of an IFN-γ response, but not peak protection. Despite delayed clearance of viral burdens, the CD8 T cell expansion and IFN-γ responses elicited in the presence of the alternative IL-12 pathway are LCMV specific because the expanded cells bind MHC tetramer molecules complexed with the LCMV epitopes NP396-404 or GP33-41, and are stimulated to express IFN-γ by viral peptides. Although the substituted IL-12 acts to promote IFN-γ production, it does not appear to be required for expansion of virus-specific CD8 T cells. It is biologically significant, however, as viral titers increase if both pathways are blocked. Thus, major contributions to adaptive CD8 T cell responses are made by innate cytokines, predominantly IFN-α/β, but alternatively IL-12, during viral infection. These innate cytokine immunoregulatory pathways can be contrasted to those characterized in response to nonviral intracellular pathogens. Similar to the Th1 responses defined under conditions of bacterial or parasitic stimuli ( 1 , 3 – 9 ), LCMV infections induce IL-2 and IFN-γ production. However, in the other microbial infections, IL-12 is the pivotal cytokine promoting Th1 responses ( 4 – 6 , 8 , 42 ). The studies presented here conclusively demonstrate that, in the complete absence of endogenous biologically active IL-12 resulting from genetic mutation of the IL-12p35 subunit, LCMV-induced CD8 T cell expansion and IFN-γ expression proceed normally . The lack of a role for IL-12 in induction of T cell IFN-γ expression confirms and extends an earlier report from this laboratory demonstrating that neutralization of endogenous IL-12 function by treatment with antibodies directed against the p40 chain does not inhibit LCMV induction of the T cell responses in IFN-α/β competent mice ( 11 ). Moreover, it is in agreement with the recent reports of lack of IL-12 effect on T cell IFN-γ responses under the conditions of treatments with antibody directed against the p40 chain during influenza virus infections ( 12 ) and genetic mutation of either the p35 or both the p35 and p40 molecules during mouse hepatitis virus infections ( 14 ). Thus, there are indications in a variety of viral infections that T cell IFN-γ responses are IL-12 independent. In addition to showing the lack of importance for IL-12 in the presence of IFN-α/β functions, however, the studies demonstrate an IFN-α/β role in supporting T cell IFN-γ production during viral infections. They define the existence of this pathway for the first time and characterize the in vivo conditions under which it is important to the host. The results are consistent with reported enhancing effects of IFN-α/β for T cell IFN-γ production under certain specific conditions in culture ( 27 – 29 , 43 ). However, the culture studies have been limited to examining the IFN-α roles for modest effects in association with IL-12 ( 28 , 43 ), CD4 T cell subset IFN-γ responses ( 27 , 43 ), and/or dramatic effects in association with the IFN-γ–inducing factor (IGIF), sometimes called IL-18 ( 29 ). Ongoing studies in our laboratory are evaluating a potential accessory role for IGIF under the conditions of viral infections. The results presented here contribute to defining the complete system by demonstrating that during viral infections IFN-α/β cytokines are dominant for T cell IFN-γ responses, mediate these effects in the absence of IL-12, and act on CD8 T cell subsets. The experiments also identify a secondary IL-12 response absent in IFN-α/βR–competent but revealed in IFN-α/βR– deficient mice . The lack of IL-12 appearance in the presence of IFN-α/β functions is consistent with the known negative regulation of IL-12 by the cytokines ( 13 ). The alternative IL-12 response is beneficial to the host because it can substitute in promoting IFN-γ production and facilitates clearance of virus. However, it is apparently sub-optimal because the conditions may fail to access the direct antiviral effects of IFN-α/β and result in delayed viral clearance. Thus, the responses elicited in the context of the IFN-α/β–mediated effects are clearly better for defense against this particular infectious agent, but the host can activate substitute defense mechanisms. Although the two divergent in vivo pathways to T cell IFN-γ are clearly established, it is not known if the IFN-α/β and IL-12 effects are mediated directly or indirectly, or at the priming as compared with the production phases. Experiments are underway examining these. Remarkably, LCMV elicits particularly high circulating levels of IFN-α/β, and under these conditions does not induce IL-12 ( 10 , 11 , 13 , 15 ). Immune responses to this infection may represent those on one end of a spectrum ranging from exclusive dependence on IFN-α/β, to codominant regulation by IFN-α/β and IL-12, to exclusive dependence on IL-12 for promoting IFN-γ production. Our hypothesis is that relative contributions would depend on presence or absence and magnitude of induction levels. MCMV and influenza infections induce more mixed responses with detectable IL-12 ( 10 – 12 , 44 ), and certain bacterial and parasitic infections also may elicit both IL-12 and IFN-α/β expression ( 6 , 45 , 46 ). Other intracellular bacteria may preferentially elicit IL-12 responses ( 4 ). Direct comparison of infections indicates that LCMV induces up to threefold higher levels and sustains longer production periods of serum IFN-α/β relative to MCMV (Cousens and Biron, unpublished results). The levels achieved during LCMV infection are sufficient to mediate significant negative regulation of IL-12 ( 13 ). Thus, the picture emerging is that the immune system is equipped to induce both IFN-α/β and IL-12 simultaneously; either will lead to conditions promoting IFN-γ production, but conditions of high IFN-α/β expression make these cytokines dominant because they also are inhibiting the IL-12 response. As stated above, such conditions appear to be beneficial because they access direct antiviral functions and are particularly conducive for induction of protective responses. Moreover, as the dramatic T cell responses to LCMV render the host more sensitive to IL-12 toxicities ( 35 , 36 ), they may additionally act to protect from detrimental immune responses. Although effects of IFN-α/β on IFN-γ production are demonstrated, the results also indicate that total and virus-specific CD8 T cell expansions are IFN-α/β independent , and that they occur even if both IFN-α/β and IL-12 functions are blocked (data not shown). Thus, other factors must be promoting CD8 T cell expansion during LCMV infections. At least one adaptive cytokine, IL-2, is apparently available to carry out this function. CM levels of IL-2 are similar with cells from IFN-α/βR KO, IL-12p35 KO, and WT mice, and only reduced by about half with cells from mice blocked in both IFN-α/β and IL-12 functions (data not shown). This cytokine is critical for CD8 T cell expansion, and as a result of supporting T cell proliferation, for peak T cell IFN-γ responses ( 17 , 21 ). Thus, the innate cytokine IFN-α/β and/or IL-12 responses and the downstream consequences of these responses do not appear to be as important as IL-2 for T cell expansion. In this regard, it has been suggested that IFN-α/β may stimulate IL-15 production to promote proliferation of memory CD8 T cells during early viral infections ( 47 , 48 ). Our results suggest that an IFN-α/β induction of IL-15 is not essential for virus-specific CD8 T cell proliferation during acute infections. However, it is interesting to note that specificities of the expanded T cells are somewhat skewed in the absence of IFN-α/β . Thus, there are additional unidentified IFN-α/β effects contributing to selection of the T cell repertoire activated against infection. The skewing of responses is observed at the level of relative proportions of CD8 T cells specifically binding tetrameric molecules complexed with NP396-404 or GP33-41 . However, as the total numbers of cells binding one or the other are similar in WT and IFN-α/βR KO mice, the magnitude of the CD8 T cell proliferative responses is IFN-α/β function independent. Nevertheless, overall responses to peptide stimulation for CD8 T cell expression of cytoplasmic IFN-γ expression , and peptide stimulation for IFN-γ production (Table I ) are reduced. Moreover, the total proportions of CD8 T cells primed to specifically respond by expressing cytoplasmic IFN-γ after ex vivo stimulation with the LCMV peptides NP396-404 or GP33-41 account for 90–95% of those sensitized to anti– CD3 stimulation during infections of WT mice, but represent only 25–60% of those sensitized during infections of IFN-α/βR KO mice . Thus, anti–CD3 reveals cells primed for T cell functions but failing to respond to the NP396-404 or GP33-41 peptides. The populations stimulated by anti–CD3, but not the tested LCMV peptides, could represent T cells having receptors (a) recognizing other LCMV epitopes, (b) nonspecifically activated, and/or (c) altered in magnitudes of functions and/or requirements for stimulation. There is evidence for the first two of these under other conditions. Although NP396-404 and GP33-41 represent the major immunodominant LCMV epitopes detected in MHC H-2 b mice ( 23 – 25 , 39 , 49 , 50 ), other minor epitopes have been identified ( 39 ). Despite prominence of the virus-specific T cell responses, “bystander” activation of memory phenotype T cells has been reported during LCMV infections ( 47 ). However, the last possibility seems most likely because CD8 T cells binding tetrameric D b GP33-41 are dramatically expanded. Further experiments are needed to conclusively distinguish between these possibilities. Nevertheless, the studies clearly document expansion of a large proportion of cells specific for LCMV epitopes and responding with IFN-γ production. LCMV is a relatively noncytopathic virus. In the absence of T cell responses and/or under specific conditions of diminishing CTL responses, sometimes called “T cell exhaustion,” persistent LCMV infections can be established. Although detectable CTL function is not induced during LCMV infection in the absence of IFN-α/β (33; data not shown), our results indicate that the conditions are sufficient for resistance and eventual viral clearance . Thus, they are in contrast to the suggestion of others that, in the absence of IFN-α/β–mediated regulation of viral replication, LCMV-induced T cell exhaustion results from an overwhelming viral burden ( 33 , 34 ). Those investigators have based their hypothesis on the lack of CTL activity without enumerating CD8 T cell numbers. In our studies, the two are dissociated; i.e., CD8 T cell expansion and IFN-γ production occur in the absence of apparent virus-specific CTL function (data not shown). However, different isolates of LCMV vary for spontaneous induction, in immunocompetent mice, of T cell exhaustion as characterized by lack of CTL ( 51 , 52 ) and persistent infection (51– 53), and it has been demonstrated that at least one of these conditions also results in the lack of virus-specific CD8 T cell expansion ( 54 ). Clearly, this is not the case under the conditions of infection in our studies. However, it is interesting to note that during chronic LCMV infections, the specificity of CD8 T cell responses can vary such that NP396-404–specific cells are deleted and functionally unresponsive GP33-41–specific cells are maintained ( 55 ), and that the skewing of specific T cells during infections of IFNα/βR KO mice is in this direction; i.e., reduced NP396-404 and increased GP33-41 specific cells . Thus, parameters in addition to the absence of IFN-α/β functions must be required to extinguish defense and establish viral persistence, but protection mediated in the absence of IFN-α/β functions may be shifting in dependence towards T cell subsets sustained for longer periods of time during infections and extended antigen stimulation. In summary, data presented here define unique divergent regulatory pathways promoting IFN-γ responses to viral infection, controlled by IFN-α/β or IL-12. They demonstrate that the strong and protective CD8 T cell responses of expansion and IFN-γ production are induced though IL-12–independent pathways during infections of immunocompetent hosts. Moreover, the studies show that in the absence of endogenous IFN-α/β, an IL-12 response can be revealed and substitute conditions to promote IFN-γ production. Although not resulting in induction of the most effective antiviral immune responses, the IL-12 substitution is beneficial. Thus, the results define uniquely IFN-α/β– controlled pathways for promoting peak defense during viral infections inducing these cytokines, and the plasticity of immune responses in accessing an alternative pathway to reach certain of the same goals. | Study | biomedical | en | 0.999999 |
10209049 | The following strains of mice were used: C57BL/6J (H-2 b ) (Thy1.2 + , Ly5.2 + ), B6.PL- Thy1 a /Cy (B6.PL; Thy1.1 + , Ly5.2 + ), B6.SJL- Ptprc a Pep3 b /BoyJ (Ly5 a ) (B6.SJL; Thy1.2 + , Ly5.1 + ), and A.BY- H2 b H2-T18 b /SnJ (ABY; Thy1.2 + , Ly5.2 + ). Mice, originally purchased from The Jackson Laboratory , were bred and housed in specific pathogen-free conditions at the Guy-Bernier Research Center according to the standards of the Canadian Committee for Animal Protection. All mice used as primary cell donors or irradiated recipients were between 8 and 20 wk of age. For 5-bromo-2′-deoxyuridine (BrdU) 1 incorporation studies, mice were given sterile drinking water containing 0.8 mg/ml BrdU ( Sigma Chemical Co. ), which was made fresh, changed daily, and protected from light. At 4–8 wk of age, mice were anesthetized by intraperitoneal injection of 75 mg/kg sodium pentobarbital (Somnotol; MTC Pharmaceuticals). Thymectomy was performed with a suction cannula introduced over the suprasternal notch. Completeness of thymectomy was verified in each animal by visual inspection at the time of killing. Cell transplantation was performed at least 2 wk after surgery. Bone marrow cells were T cell depleted and transplanted as previously described ( 23 ). Recipient mice received 10 Gy total body irradiation on day 0, the day of transplant. Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement. Efficacy of depletion was assessed by flow cytometry. Spleen or axillary, cervical, and inguinal LN cells were harvested and washed. The number of T cells injected was determined by flow cytometry using an anti-Thy1.1 or anti-Thy1.2 Ab. Bone marrow and spleen or LN cells were given as a single intravenous injection, via the tail vein, in a volume of 0.5 ml. The following Abs were obtained from PharMingen : biotinylated anti-CD8α (53-6.7; rat IgG2a) detected with Cy-chrome™–streptavidin, FITC-conjugated anti–TCR-α/β (H57-597; hamster IgG), anti-Vβ3 (KJ25; hamster IgG), anti-Vβ5.1,2 (MR9-4; mouse IgG1), anti-Vβ6 (RR4-7; rat IgG2b), anti-Vβ7 (TR310; rat IgG2b), anti-Vβ8.1,2 (MR5-2; mouse IgG2a), anti-Vβ9 (MR10-2; mouse IgG1), anti-Vβ10 b (B21.5; rat IgG2a), anti-Vβ11 (RR3-15; rat IgG2b), anti-Vβ13 (MR12-3; mouse IgG1), anti-Vβ14 (14-2; rat IgM), anti-Vβ17 a (KJ23; mouse IgG2a), anti-CD45.1 (Ly5.1; 104; mouse IgG2a), anti-CD45.2 (Ly5.2; A20; mouse IgG2a); PE-conjugated anti-Thy1.2 (30-H12; rat IgG2b), anti-Thy1.1 (OX-7, mouse IgG1,k), anti-CD62L (MEL-14; rat IgG2a,k), anti–TCR-γ/δ (GL3; hamster IgG), and specific Cy-chrome™-conjugated anti-CD4 (RM4-5; rat IgG2a), anti-CD8α (53-6.7; rat IgG2a) Abs, and their isotypic controls. PE-conjugated anti-CD4 (YTS 191.1, rat IgG2b), anti-CD8α (YTS 169.4, rat IgG2b) Abs and their isotypic controls were purchased from Cedarlane Labs., and the FITC-conjugated anti-BrdU Ab was purchased from Becton Dickinson . Cell surface staining was performed as previously described ( 23 ). BrdU labeling was performed as described by Tough and Sprent ( 27 ). In brief, after surface staining, cells were resuspended in cold 0.15 M NaCl, fixed by dropwise addition of cold 95% ethanol, incubated for 30 min on ice, and washed with PBS. The cells were then incubated with PBS containing 1% paraformaldehyde and 0.01% Tween 20 for 30 min, pelleted, and then incubated for 30 min with 50 KU of DNase I ( Sigma Chemical Co. ) in 0.15 M NaCl and 4.2 mM MgCl 2 , pH 5. After washing, cells were incubated with FITC-conjugated anti-BrdU for 30 min and washed. Cells were analyzed on a FACScalibur ® using the CellQuest program or on a FACScan ® using the Lysis II program (all from Becton Dickinson ). Irradiated euthymic or thymectomized A.BY (Thy1.2, Ly5.2) recipients received a graft containing B6.PL (Thy1.1, Ly5.2) bone marrow cells (as a source of hematopoietic progenitors) with or without mature T cells harvested from the LNs of B6.SJL donors (Thy1.2, Ly5.1). The origin of recipient T cells was determined according to their Thy1/ Ly5 phenotype. Recipients were studied on day 100 after transplant; i.e., after the allogeneic acute phase of GVHD was terminated. We found that two factors affected the level of T cell reconstitution: the presence/absence of a host thymus and the presence/absence of GVHD . Indeed, based on the number of both CD4 + and CD8 + T cells found in the spleen of day 100 chimeras, the following hierarchy was observed : thymus + GVHD − (group A) > thymus − GVHD − (group B) > thymus + GVHD + (group C) > thymus − GVHD + (group D). These results confirm that, in thymectomized recipients of a T cell–depleted graft, some extrathymic differentiation of donor hematopoietic progenitors may take place, but that this cannot compensate for the absence of the classical thymic differentiation pathway (group B versus A) ( 22 , 23 , 28 , 29 , 53 ). More importantly, they show that, in terms of T cell reconstitution of secondary lymphoid organs, the impact of GVHD (groups C and D) is even more deleterious than that caused by the mere absence of thymus (group B). In GVHD + euthymic recipients grafted with low or high numbers of donor T cells (group C), the number of spleen T cells was decreased 6–12-fold relative to recipients without GVHD (group A). T cell hypoplasia was slightly more severe when the number of grafted T cells was increased from 0.4 to 1.6 × 10 6 . Interestingly, most T cells found in euthymic GVHD + recipients (group C) did not derive from expansion of grafted postthymic T cells, but rather from the donor hematopoietic stem cells. We could ascertain that the vast majority of these T lymphocytes originated from donor hematopoietic progenitors that had differentiated in the GVHD + thymus and not in extrathymic sites, because their numbers were decreased eightfold in athymic GVHD + recipients (group D versus C). In athymic hosts, the occurrence of GVHD (group D) caused an extremely severe T cell hypoplasia with T cell numbers decreased by fivefold compared with athymic GVHD − recipients (group B). Among the few T cells that were found in athymic GVHD + mice, ∼55% were derived from grafted mature T cells, while ∼45% had the phenotype of donor hematopoietic stem cells and were likely derived from the extrathymic differentiation of these progenitors. T cells derived from grafted postthymic T cells were much less abundant (∼14-fold) in athymic GVHD + hosts (group D) than what we previously observed in similarly treated syngeneic (GVHD − ) recipients ( 23 ). Two main conclusions can be drawn from these results. First, GVHD not only impairs reconstitution via the thymic pathway, but also abrogates expansion of grafted postthymic T cells. Second, although thymus function is impaired in GVHD + chimeras, intra-thymic maturation still represents the most effective pathway for T cell reconstitution in these mice. Results from Fig. 1 indicate that in GVHD + mice, the ability of thymic-independent pathways to restore peripheral T cell pools is extremely poor and much inferior to that of the classical thymic pathway. Besides these quantitative considerations, we were interested in determining whether the different origin of T lymphocytes in athymic versus euthymic GVHD + chimeras would have any impact on their Vβ profile. This question was addressed because a continuous thymic output can contribute to the maintenance of T cell diversity, whereas the repertoire of T cell pools that rely solely on the expansion of postthymic T cells is more prone to skewing after stochastic encounter with antigens ( 38 , 54 ). Thus, we evaluated by flow cytometry the TCR Vβ profile of T cells (Thy1.1 + ) that differentiated in the thymus of nonthymectomized GVHD + recipients, of (Thy1.2 + ) cells that originated from the expansion of grafted mature T cells in athymic GVHD + recipients, and of age-matched A.BY controls. In both controls and euthymic GVHD + mice, the proportion of CD4 + and CD8 + cells bearing various Vβ elements was remarkably constant and, except for Vβ11 + cells, showed very little variation from mouse to mouse . In contrast, considerable variability in the usage of Vβ chains was found in athymic GVHD + mice . Thus, among CD8 + lymphocytes, the percentage of cells expressing Vβ5 or Vβ6 elements was 14–16 and 7–7.5%, respectively, in euthymic GVHD + mice, but 4–20 and 2–14.5%, respectively, in athymic GVHD + mice. Analyses based on size heterogeneity or on sequence of the CDR3 region will be required to assess more precisely the diversity and clonality of these T cell populations ( 55 , 56 ). Nevertheless, our results indicate that the TCR repertoire of T cells derived from the expansion of grafted mature T cells is subject to dramatic skewing in athymic GVHD + mice. The absence of Vβ skewing in euthymic GVHD + mice suggest that, even though their thymus is damaged, it has the ability to maintain a Vβ profile that is similar to that of age-matched controls. To address the biological significance of the residual thymus function detected in nonthymectomized GVHD + mice, we followed up to day 95 posttransplant the survival of mice from experimental groups presented in Fig. 1 . No deaths were observed either in thymectomized or nonthymectomized GVHD − recipients. In contrast, the presence/absence of the thymus significantly influenced the survival of GVHD + mice. Indeed, during the 100-d observation period, death rates were greater in thymectomized than in nonthymectomized hosts: 25 vs. 0% and 65 vs. 10% for thymectomized versus nonthymectomized recipients grafted with 0.4 and 1.6 × 10 6 donor T cells, respectively. Thus, at least in mice housed under specific pathogen-free conditions, the limited T cell reconstitution afforded by the GVHD + thymus has a major impact on posttransplant survival. Having found that practically all T cells found in day 100 GVHD + mice derived from intra-thymic differentiation of donor hematopoietic stem cells, we wanted to quantitatively assess the thymic function of GVHD + mice. Relative to normal mice, thymus cellularity was decreased by 33–50% in GVHD + mice transplanted with a low or high number of T cells . The proportion of immature double- versus single-positive CD4 + and CD8 + thymocytes was similar in GVHD + versus normal thymi . To get a better appraisal of thymus function, we measured the rate of production of recent thymic emigrants after in vivo BrdU labeling ( 27 ). Euthymic A.BY recipients of B6.PL bone marrow cells with or without 0.4 × 10 6 B6.SJL LN T cells were given BrdU-supplemented drinking water for 21 d beginning on day 70 posttransplant, and three-color flow cytometry analyses were performed selectively on CD62L + T cells. For both the CD4 + and the CD8 + subsets, recent thymic emigrants were defined as CD62L + BrdU lo cells. The CD62L − T cell subset contains antigen-experienced activated/memory T cells, while the CD62L + subset contains practically all naïve T cells and some “revertant” antigen-experienced T cells ( 27 , 57 ). The intensity of BrdU labeling provides a convenient way to distinguish recent thymic emigrants (BrdU lo ) from “older” peripheral T cells (BrdU hi ) that divide during the BrdU-labeling period. Low BrdU labeling of recent thymic emigrants results from cold target competition in the thymus ( 27 ). Indeed, high levels of apoptosis with local breakdown of DNA, as found in the thymus but not in secondary lymphoid organs, leads to cold target competition for BrdU incorporation. After administration of BrdU for 21 d, the total number of recent thymic emigrants (CD4 + /CD62L + /BrdU lo and CD8 + /CD62L + /BrdU lo ) per spleen was fourfold lower in GVHD + hosts relative to normal mice and GVHD − recipients . Thus, thymus output in GVHD + mice was significantly decreased but not absent. Interestingly, for both CD4 + and CD8 + T cells, the percentage of CD62L + elements that were BrdU lo was similar in mice with or without GVHD . This suggests that the reduced size of the CD62L + compartment, which encompasses naïve and revertant T cells, cannot be wholly ascribed to a decreased input of recent thymic emigrants. If this were the case, as in senescent individuals, one would expect not only the absolute number but also the proportion of BrdU lo elements among CD62L + cells to be decreased ( 58 ). Results presented in Fig. 1 showed that the mere absence of thymus (group B) does not entail the severe level of lymphoid hypoplasia found in GVHD + mice (groups C and D). In addition, we found that the thymus of GVHD + mice was still functional with a thymocyte production equivalent to 25% that of 8-wk-old controls or 18-wk-old GVHD − recipients . Such a level of thymus export has been shown to be sufficient to sustain the size of T cell compartments in secondary lymphoid organs of otherwise normal senescent mice ( 39 , 40 ). Thus, thymus hypoplasia cannot be held solely responsible for GVHD- associated T cell hypoplasia. These data point to the existence of other factors that perturb peripheral homeostasis of T cell compartments in GVHD + mice. To evaluate the rate of division of peripheral T cells, BrdU labeling was performed for 21 d, as in the previous experiment. Results for CD62L + and CD62L − subsets were analyzed separately because it has been shown that CD62L − cells divide more rapidly than CD62L + cells ( 27 ), and the proportion of CD62L + versus CD62L − T cells were different in our experimental groups . Strikingly, the kinetics of BrdU labeling was similar in GVHD − recipients, GVHD + hosts and normal controls . This suggests that the reduced size of the CD62L + and CD62L − peripheral compartments is not due to a proliferative defect of resident T cells, otherwise the proportion of dividing (BrdU + ) T cells would have been decreased. Since BrdU is not reused, it was possible to perform pulse–chase experiments. Thus, after being placed on BrdU water for 21 d, mice were transferred to normal water to examine the rate of decay of BrdU-labeled cells up to day 75. This approach has been used notably to show that the peripheral T cell lymphopenia of BB rats is caused by a shortened survival of T cells associated with an increased apoptotic death rate relative to WF rat controls ( 59 ). The intensity of BrdU labeling in T cells from GVHD + and control mice on days 21 and 75 is depicted in Fig. 6 . The rate of disappearance of BrdU-labeled T cells was similar in the three experimental groups so that, by day 75, the remaining proportion of BrdU-labeled cells was ∼20% in normal mice, GVHD − recipients, and GVHD + hosts . We deducted from the above BrdU-labeling experiments that the failure of peripheral homeostatic mechanisms to compensate for the moderate decrease in thymic output and to prevent the severe T cell depletion in GVHD + mice could neither be ascribed to an impairment of T cell proliferative activity , nor to a shortened half-life of BrdU-labeled cells . These findings suggest that the deficient expansion of the postthymic T cell compartment in GVHD + mice was not due to an intrinsic lymphocyte defect but to an extrinsic microenvironment abnormality. This led us to raise the intriguing possibility that the number of peripheral T cell niches might be decreased in GVHD + mice. This premise, according to which T cell hypoplasia would represent a problem of soil (environment) rather than seed (lymphocytes), entails two crucial corollaries: (a) T cells from GVHD + mice should expand normally in normal mice, and (b) lymphoid hypoplasia of GVHD + mice should not be corrected by infusion of (host-tolerant) T cells from normal mice. To determine whether T cells from GVHD + mice would expand in normal mice, we compared the expansion of T cells from GVHD + mice with that of normal T cells after injection in normal thymectomized/irradiated recipients (with no defect in peripheral niches). Thus, thymectomized/irradiated A.BY or B6.SJL recipients were transplanted with 2 × 10 6 Thy1.1 + Ly5.2 + T cells harvested from the spleen of nonthymectomized GVHD + mice and 10 7 T cell– depleted C57BL/6 bone marrow cells. The expansion of T cells from GVHD + mice was compared with that of normal T cells 30 d after injection (together with T cell–depleted bone marrow cells) into thymectomized/irradiated syngeneic recipients . In all recipients, the origin of T lymphocytes could be determined according to their Thy1/Ly5 phenotype. Strikingly, the level of T cell reconstitution found in syngeneic (B6.SJL) or allogeneic (A.BY) secondary recipients of GVHD + T cells was similar to that of hosts repopulated with normal T cells . The numbers of T cells harvested from secondary recipients were increased twofold relative to those in day 100 GVHD + mice. This difference is impressive when one considers that (a) the T cell reconstitution of normal secondary hosts was achieved after only 30 d (versus 100 d in GVHD + mice), and (b) GVHD + mice had a measurable thymic output, whereas reconstitution of thymectomized secondary hosts was achieved strictly by expansion of transplanted postthymic T cells. Hence, T cells from GVHD + mice expanded normally when transferred to normal hosts. As suggested by the studies presented in Fig. 5 , this shows that the failure of T cells to expand in GVHD + mice is not due to an intrinsic T cell–proliferative defect. Interestingly, the fact that normal reconstitution was achieved not only in syngeneic (B6.SJL) but also in allogeneic (A.BY) secondary hosts indicates that T cells found in the spleen of day 70 GVHD + mice could not elicit GVHD, and thus were purged of functional host-reactive T cells. As these T cells had differentiated in the thymus of the GVHD + mice, this observation supports the concept that, at least after the acute phase of GVHD, the thymus of GVHD + mice efficiently performs negative selection of host-reactive thymocytes ( 60 ). Furthermore, the lack of antihost alloreactive T cells in day 70 GVHD + mice argues against the possibility that a persistent GVHD activity per se could be responsible, notably via production of some cytokine(s), for the persistent T cell hypoplasia found in long-term (day 100) chimeras. We next evaluated whether adoptive transfer of normal postthymic T cells would correct the T cell lymphopenia of GVHD + mice. The fate of normal T cells after passive transfer to histocompatible recipients depends on the quantity of available T cell niches. When the size of the peripheral T cell compartment is normal, such that few T cell niches are available, most donor T cells disappear soon after transfer ( 61 ). In contrast, transfer of T cells to “T-less” recipients, in which numerous empty niches are available, is followed by the persistence of a large proportion of donor-derived T cells and a considerable antigen-driven expansion of these cells, which results in the restoration of the size of the peripheral compartment ( 26 , 62 ). To obtain T cells of B6 origin that were tolerant to A.BY antigens, normal irradiated A.BY mice were transplanted with 10 7 T cell–depleted C57BL/6 bone marrow cells. 60 d later, a spleen cell suspension from these chimeras containing 5 × 10 6 Thy1.2 + Ly5.2 + T cells (of C57BL/6 origin) was injected into day 60 GVHD + secondary hosts, and the fate of Thy1.2 + Ly5.2 + T cells was assessed 40 d after transfer. Before transfer, no Thy1.2 + Ly5.2 + cells were present in GVHD + secondary hosts that had been constructed by injection of 10 7 B6.PL bone marrow cells + 0.4 × 10 6 B6.SJL LN T cells into irradiated nonthymectomized A.BY hosts. As a positive control, the proliferative potential of postthymic T cells transferred into secondary hosts was evaluated in a group of irradiated/thymectomized GVHD − recipients. When transferred into GVHD − secondary hosts, postthymic T cells showed a major expansion , whose magnitude was similar to what has been reported after transfer into irradiated/thymectomized syngeneic recipients ( 23 ). In contrast, transfer of host-tolerant C57BL/6 T cells did not increase the size of the peripheral T cell pool of GVHD + recipients . Indeed, 40 d after their transfer into GVHD + secondary hosts, only trace amounts of Thy1.2 + Ly5.2 + T cells were recovered. Thus, supply of postthymic T cells that had differentiated in a normal GVHD − thymus and possessed a normal proliferation potential had no influence on the size of the splenic T cell pool in GVHD + mice. Together with those presented in Fig. 8 , these results demonstrate that the perturbed homeostasis of the peripheral T cell pool in GVHD + mice is not due to a lymphocyte intrinsic anomaly, but rather to a failure of the peripheral environment to support the seeding and/or expansion of postthymic T cells. In conclusion, the results presented in this paper show that both central and peripheral mechanisms contribute to the long lasting T cell hypoplasia found in GVHD + mice. GVHD + mice have a decreased thymic output, but this defect cannot be held solely responsible for peripheral T cell hypoplasia. The lack of reconstitution of normal peripheral T cell compartments is largely accounted for by an inability of postthymic T cells to expand in the secondary lymphoid organs of GVHD + hosts. Collectively, our results provide convincing evidence that the failure of mature T cells to expand in GVHD + mice is not due to an intrinsic lymphocyte defect, but to an undefined abnormality of the lymphoid microenvironment: (a) postthymic T cells from GVHD + mice expanded normally in syngeneic or allogeneic secondary hosts, and (b) normal T cells failed to expand in GVHD + mice. Until it is better defined, we think that this microenvironment defect is most consistent with a decrease in the number of functional peripheral T cell niches. As mentioned in the introduction, resident dendritic cells may represent the most crucial elements of these niches. Thus, the possible influence of quantitative and/or qualitative (e.g., expression of MHC molecules, chemokines, cytokines) dendritic cell defects on T cell homeostasis in GVHD deserves further investigation. To our knowledge, these questions have not been addressed in mouse models of GVHD. However, according to the limited information available from human studies, it is noteworthy that chronic GVHD appears to be associated with a major decrease in the numbers of dendritic cells in the skin and secondary lymphoid organs ( 63 – 65 ). Alternatively, we cannot discard the possibility that the microenvironment defect epitomized herein as a reduction of functional T cell niches could be related to the presence of a toxic and/or absence of supportive soluble factor produced by cells other than dendritic cells. Nevertheless, two elements argue against some involvement of an inhibitory T cell–derived cytokine: no GVHD-inducing T cells were detected in our day 100 GVHD + mice, and the defect was not transferred to secondary recipients of T cells from GVHD + mice . These considerations emphasize the need for a precise definition of the peripheral T cell niches, whose number likely dictates the size of peripheral T cell pools. Furthermore, it will be important to determine whether a similar microenvironment defect could represent an unrecognized cause of acquired immunodeficiency in other settings. For example, damage to peripheral T cell niches could provide a plausible explanation for the prolonged hypoplasia of secondary lymphoid organs observed after massive T cell responses ( 66 , 67 ). Importantly, a loss of peripheral T cell niches must not be considered irreversible a priori, and may possibly be amenable to therapy. Indeed, recent studies in RIP-LT transgenic mice have demonstrated that engineered local release of lymphotoxin can trigger lymphoid neogenesis characterized by the formation of well organized and functional “tertiary” lymphoid tissue ( 68 , 69 ). | Study | biomedical | en | 0.999996 |
10209050 | Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ). The mAbs against the four TRAIL receptors (M271, IgG2a anti–TRAIL-R1; M413, IgG1 anti–TRAIL-R2; M430, IgG1 anti–TRAIL-R3; and M444, IgG1 anti–TRAIL-R4) were produced at Immunex by immunizing BALB/c mice (The Jackson Laboratory ) with a purified fusion protein consisting of the extracellular domain of human TRAIL-R1, -R2, -R3, or -R4 coupled to the constant region of human IgG1 (huTRAIL-R:Fc) in Titermax ( CytRx Corporation ). Mice were boosted three times, and spleen cells were fused with the murine myeloma NS1 in the presence of 50% polyethylene glycol in PBS followed by culture in DMEM/HAT and DMEM/HT selective media. Supernatants from positive wells were tested for the ability to bind the appropriate TRAIL receptor in an ELISA (cell-based ELISA using CV1 cells transfected with TRAIL receptor cDNA) and reactivity to huTRAIL-R:Fc in Western blots. Hybridomas that produced antibodies that bound to huTRAIL-R:Fc, but not human IgG1, were cloned by three rounds of limiting dilution. All mAbs were purified by protein A affinity chromatography. The matrix metalloproteinase inhibitors, TAPI (TNF protease inhibitor ) and KB8301, were obtained from Immunex and PharMingen , respectively. The soluble fusion proteins TRAIL-R2:Fc, Fas:Fc, and TNFR:Fc were produced at Immunex . The leucine zipper (LZ)-TRAIL expression plasmid ( 34 ) and the production and purification of LZ-huTRAIL ( 40 ) have been described previously. The ovarian carcinoma cell line (OVCAR3) was obtained from Dr. Richard F. Camalier, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, MD). The human melanoma cell lines (WM 793 and 164) were obtained from Dr. M. Herlyn, Wistar Institute (Philadelphia, PA). The human prostate carcinoma cell line (PC-3) was obtained from Dr. Michael Cohen, University of Iowa (Iowa City, IA). The human colon carcinoma cell line (Colo205) and the human lung adenocarcinoma cell line were provided by Dr. Brian Gliniak and Tim Lofton, respectively ( Immunex ). L929 cells and normal human foreskin fibroblasts were obtained from American Type Culture Collection. All tumor cell lines were cultured as directed. The normal human lung fibroblasts were purchased from Clonetics Corporation and cultured as directed. Peripheral blood Mφ were enriched using countercurrent elutriation. Cells from leukopheresis packs obtained from healthy volunteers were loaded onto a JE-5 elutriator (Beckman), and 50-ml fractions were collected while increasing the flow rate from 65 to 85 ml/min at 2,000 rpm. Mφ-enriched fractions generated from a flow rate >75 ml/min were >90% CD14 + as assessed by flow cytometric analysis using TUK-4, an IgG2a anti-CD14 (Caltag Laboratories, Inc.). Untreated or cytokine-stimulated Mφ were incubated with the following unlabeled primary mAbs for 1 h at 4°C: MOPC-21, MOPC-173, M181, M271, M413, M430, M444, mAb11, and NOK-1. After three washes, primary antibody binding was detected with a PE-conjugated, Fc-specific, mouse anti–human F(ab′) 2 (Jackson ImmunoResearch Laboratories). Staining for TNF was done in the presence of the matrix metalloproteinase inhibitor, TAPI (50 μM). Staining for FasL was done in the presence of the matrix metalloproteinase inhibitor, KB8301 (10 μM). Cells were analyzed immediately after staining or fixed in 1% paraformaldehyde until analysis on a FACSCalibur™ ( Becton Dickinson ). Mφ were cultured for 12 h in medium alone, GM-CSF, IFN-γ, or IFN-α, washed, and resuspended in complete medium. Tumor cells were labeled with 100 μCi of 51 Cr for 1 h at 37°C, washed three times, and resuspended in complete medium. To determine TRAIL- induced death, 51 Cr-labeled tumor cells (10 4 /well) were incubated with varying numbers of Mφ effector cells for 8 h. As a positive control, soluble LZ-TRAIL was added to the target cells at the indicated concentrations. In some cultures, TRAIL-R2:Fc, Fas:Fc, or TNFR:Fc (20 μg/ml) was added to the Mφ effector cells 15 min before adding tumor cell targets. All cytotoxicity assays were performed in round-bottomed 96-well plates, and the percent specific lysis was calculated as: 100 × (experimental cpm − spontaneous cpm)/(total cpm − spontaneous cpm). Spontaneous and total release were determined in the presence of either medium alone or 1% NP-40, respectively. The presence of TRAIL-R2:Fc, Fas:Fc, or TNFR:Fc during the assay had no effect on the level of spontaneous release of 51 Cr by the target cells. For analysis of tumor cell apoptosis, tumor cell targets were incubated with unstimulated or cytokine-stimulated Mφ as described above. Apoptotic cell death of the tumor cells was measured by flow cytometry using FITC-conjugated annexin V and propidium iodide (Apoptosis detection kit; R&D Systems) as per the manufacturer's protocol. Light scatter characteristics were used to identify the tumor cells. The specific inhibitor of NO synthase, N G -monomethyl- l -arginine (L-NMMA; AerBio, Ltd.), was used to block Mφ NO production ( 44 ). Mφ were stimulated with the appropriate cytokine and then incubated with the tumor cell targets as above. Mφ were cultured in the presence of 300 μM L-NMMA at all steps throughout the assay. The presence of L-NMMA had no effect on the level of spontaneous release of 51 Cr by the target cells during the assay. Mφ were cultured for 2 or 12 h in medium alone or LPS (5 ng/ml), washed, and resuspended in complete medium. L929 cells were labeled with 100 μCi of 51 Cr for 1 h at 37°C, washed three times, and resuspended in complete medium. To determine TNF-induced death, 51 Cr-labeled L929 cells (10 4 /well) were incubated with varying numbers of Mφ effector cells for 8 h. As a positive control, soluble TNF was added to the target cells at the indicated concentrations. In some cultures, TNFR:Fc or TRAIL-R2:Fc (20 μg/ml) was added to the Mφ effector cells 15 min before adding tumor cell targets. L929 cytotoxicity assays were performed in an identical manner as for the human tumor cell lines. The presence of TNFR:Fc or TRAIL-R2:Fc during the assay had no effect on the level of spontaneous release of 51 Cr by the L929 target cells. Mφ were cultured for 12 h in medium alone or with cytokine (IL-1, IL-2, IL-3, IL-4, IL-7, IL-10, IL-12, IL-15, GM-CSF, or IFN-γ; all cytokines were used at 100 ng/ml), and then labeled with 100 μCi of 51 Cr for 1 h at 37°C, washed three times, and resuspended in complete medium. To determine sensitivity to TRAIL-induced death, 51 Cr-labeled Mφ (10 6 /well) were incubated with LZ-TRAIL for 8 h. Assays were performed as described above. Total RNA was isolated from Mφ with TRIzol reagent (Life Technologies) as per the manufacturer's instructions. RNA samples (1 μg each) were tested for DNA contamination by 30 cycles of PCR with human β-actin primers. After it was shown that there was no DNA contamination, cDNA synthesis was performed using an RNA PCR kit ( Perkin-Elmer ) with the supplied oligo d(T) 16 primer. Reverse transcription was performed using a thermal program of 25°C for 10 min, 42°C for 30 min, and 95°C for 5 min. PCR reactions were performed using the following primers: human β-actin (forward: 5′-GAAACTACCTTCAACTCCATC-3′; reverse: 5′-CGAGGCCAGGATGGAGCCGCC-3′); human TRAIL-R1 (forward: 5′-CTGAGCAA-CGCAGACTCGCTGTCCAC-3′; reverse: 5′-TCCAAGGACA-CGGCAGAGCCTGTGCCAT-3′); human TRAIL-R2 (forward: 5′-GCCTCATGGACAATGAGATAAAGGTGGCT-3′; reverse: 5′-CCAAATCTCAAAGTACGCACAAACGG-3′); human TRAIL-R3 (forward: 5′-GAAGAATTTGGTGCCAATGCCACTG-3′; reverse: 5′-CTCTTGGACTTGGCTGGGAGATGTG-3′); human TRAIL-R4 (forward: 5′-CTTTTCCGGCGGCGTTCATGTCCTTC-3′; reverse: 5′-GTTTCTTCCAGGCTGCTTCCCTTTGTAG-3′); and human TRAIL (forward: 5′-CAACTCCGTCAGCTCGTTAGAAAG-3′; reverse: 5′-TTAGACCAACAACTATTTCTAGCACT-3′), giving products of 219, 506, 502, 612, 453, and 443 bp, respectively. β-actin PCR cycle conditions were 95°C for 45 s, 55°C for 1 min, and 72°C for 45 s for 30 cycles. TRAIL-R1, -R2, and -R3 cycle conditions were 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 30 cycles. TRAIL-R4 cycle conditions were 95°C for 4 min 15 s, followed by 30 cycles of 95°C for 45 s, 60°C for 45 s, and 72°C for 45 s. TRAIL cycle conditions were 95°C for 45 s, 55°C for 45 s, and 72°C for 45 s for 30 cycles. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide. To compare the difference in TRAIL, FasL, and TNF surface expression, peripheral blood human Mφ were isolated and cultured with several molecules known to induce Mφ activation and differentiation (IFN-γ, IFN-α, GM-CSF, and LPS), and then examined by flow cytometry. Significant TRAIL expression was detected on the Mφ cultured for 12 h in the presence of either IFN-γ or IFN-α, but not with either GM-CSF or LPS . In contrast, FasL expression on Mφ was undetectable after either 2- or 12-h stimulation with any of the cytokines or LPS in the presence of the metalloproteinase inhibitor, KB8301 . Analysis of the surface levels of TNF demonstrated no measurable increase after stimulation with IFN-γ or IFN-α; however, stimulation with LPS for 2 h led to increased surface levels of TNF that disappeared by 12 h . To inhibit the cleavage of membrane TNF, Mφ were cultured in the presence of the metalloproteinase inhibitor, TAPI (TNF protease inhibitor ). These data demonstrate that TRAIL, but not FasL and TNF, is induced after IFN stimulation, and that the expression of TRAIL and TNF on Mφ is regulated by distinct activation stimuli and with different kinetics. The results from Fig. 1 demonstrate that Mφ stimulated with IFN upregulate the expression of TRAIL on the cell surface. Thus, to examine the functional activity of TRAIL in this setting, Mφ were stimulated with either GM-CSF, IFN-γ, or IFN-α for 12 h and then cultured in the presence of OVCAR3, a TRAIL-sensitive human ovarian carcinoma cell line. Although the unstimulated or GM-CSF–treated Mφ demonstrated minimal tumoricidal activity toward OVCAR3, the Mφ stimulated with IFN-γ or IFN-α were potent killers of these TRAIL-sensitive tumor cells over a broad range of E/T ratios . A titration of both IFN-γ and IFN-α concentrations revealed that as little as 10 pg/ml led to enhanced Mφ cytotoxicity against the tumor cells; however, GM-CSF did not induce any antitumor activity at any concentration tested (100 ng/ml to 10 pg/ml; data not shown). Moreover, the IFN-stimulated Mφ were as effective in killing the tumor target cells as recombinant, soluble TRAIL (LZ-TRAIL ). The tumoricidal activity of IFN-stimulated Mφ was also examined on a TRAIL-resistant human melanoma cell line, WM 164, and a TRAIL-sensitive human melanoma cell line, WM 793 ( 38 ). The TRAIL-resistant melanoma (WM 164) was also resistant to the Mφ-mediated cytotoxicity, whereas the TRAIL-sensitive melanoma (WM 793) was quite sensitive to the cytotoxic activity of either IFN-γ– or IFN-α–stimulated Mφ . The tumoricidal activity of both IFN-γ– or IFN-α–stimulated Mφ was seen from multiple donors and with other TRAIL-sensitive tumor cells (Table I ). Two normal human fibroblast cell types were also tested for sensitivity to the cytokine-stimulated Mφ and were found to be resistant in all conditions from multiple donors. Although FasL was not detected on the surface of the activated Mφ , activated Mφ have been shown to release soluble FasL from intracellular stores ( 25 ). Therefore, to confirm that the observed tumoricidal activity was specific to TRAIL and not FasL, IFN-γ–stimulated Mφ were pretreated with either TRAIL-R2:Fc ( 35 ) or Fas:Fc before adding the tumor cell targets. The TRAIL-R2:Fc reduced target cell death to control (unstimulated Mφ effector) levels, whereas Fas:Fc did not alter the ability of the IFN-γ–treated Mφ to mediate tumor lysis . Finally, to determine whether Mφ NO production contributed to the measured cytotoxic activity, Mφ were stimulated as above but in the absence or presence of the NO synthase inhibitor, L-NMMA ( 44 ). The cytotoxic activity of the IFN-γ– and IFN-α–stimulated Mφ was not decreased in the presence of L-NMMA compared with Mφ stimulated in the absence of the inhibitor . Similar results were observed with other tumor cell targets (data not shown). Furthermore, analysis of NO production by the Mφ after 12 h stimulation, as measured by the accumulation of nitrite, revealed no increase in nitrite levels in the culture supernatants with any of the different stimuli compared with unstimulated Mφ (data not shown). Collectively, these results confirm that the TRAIL expressed on Mφ mediates the killing of tumor cells, demonstrating a novel mechanism of Mφ-mediated apoptosis. Although the release of 51 Cr from the tumor cell targets as measured in Fig. 2 indicates the amount of cell death, it does not discriminate between apoptotic and necrotic cell death. Previous reports have demonstrated that TRAIL-induced cell death occurs through an apoptotic mechanism ( 30 , 34 , 38 , 41 ). To confirm that the tumor cell death induced by the IFN-stimulated Mφ was mediated through an apoptotic mechanism, the binding of FITC-conjugated annexin V to the tumor cells was analyzed. Annexin V preferentially binds to phosphatidylserine, a phospholipid component of the inner leaflet of the plasma membrane that is rapidly externalized during apoptosis ( 45 , 46 ). Upon staining the OVCAR3 tumor cells after 6 h incubation with unstimulated or cytokine-stimulated Mφ (E/T ratio 2:1) or soluble LZ-TRAIL, only those tumor cells incubated with IFN-stimulated Mφ or LZ-TRAIL were positive for FITC-annexin V binding , indicating that these cells were dying from the induction of apoptosis. Morphological changes (membrane blebbing and release of apoptotic bodies) were also observed using light microscopy (data not shown). Having demonstrated that IFN-stimulated Mφ express functional TRAIL, the differences between TRAIL and TNF-mediated tumoricidal activity by Mφ were examined. The results presented in Fig. 1 show that TNF was only expressed on Mφ after incubation with LPS. Thus, to demonstrate the biologic activity of the cell surface TNF detected after LPS stimulation, 2- and 12-h LPS-stimulated Mφ were evaluated for the ability to kill the TNF-sensitive target cell, L929 ( 17 ). In direct correlation with the flow cytometric results, the 2-h LPS-stimulated Mφ, but not the 12-h LPS-stimulated Mφ, killed L929 target cells to levels comparable to soluble TNF . When tested for sensitivity to LZ-TRAIL, the L929 cells were found to be resistant (data not shown). This killing was TNF-specific as demonstrated by the significant inhibition of the lysis of L929 cells upon the addition of TNFR:Fc, but not TRAIL-R2:Fc . To further demonstrate that the cytotoxic activity of IFN-γ–stimulated Mφ was mediated by TRAIL and not TNF, tumor cell lysis was measured in the presence of TRAIL-R2:Fc or TNFR:Fc. Only TRAIL-R2:Fc, and not TNFR: Fc, inhibited the IFN-γ–stimulated Mφ from killing the tumor cell targets . Thus, human Mφ have multiple mechanisms for killing a variety of target cells depending on the activation mechanism. Because TRAIL can interact with two death-inducing and two non-death-inducing receptors, the distribution of the four known TRAIL receptors on the Mφ surface using receptor-specific mAbs was investigated. Unstimulated Mφ expressed both TRAIL-R2 and -R3, whereas the levels of TRAIL-R1 and -R4 were at or below detection . However, mRNA for each of the four TRAIL receptors could be detected by reverse transcription (RT)- PCR analysis . The kinetics of TRAIL, TRAIL-R2, and TRAIL-R3 expression after IFN-γ stimulation were then measured at both the protein level by flow cytometry and the mRNA level by RT-PCR. Increased TRAIL expression could be detected by 2 h on the cell surface after addition of IFN-γ , whereas RT-PCR analysis demonstrated that TRAIL mRNA levels increased by 1 h . No consistent change in TRAIL protein or mRNA levels was detected after GM-CSF treatment compared with untreated Mφ . Examination of the surface levels of TRAIL-R2 and -R3 during this same 8-h period revealed that IFN-γ–stimulated Mφ downregulated TRAIL-R2 expression, whereas TRAIL-R3 was only slightly downmodulated . In contrast, incubation with GM-CSF for 8 h resulted in a slight increase in TRAIL-R2 expression. Analysis of mRNA from IFN-γ–stimulated Mφ revealed that TRAIL-R2 mRNA levels remained relatively constant over the 8-h period, whereas the TRAIL-R2 mRNA levels increased in Mφ stimulated with GM-CSF over time . No changes in TRAIL-R3 mRNA were observed with IFN-γ or GM-CSF incubation during this period of time , nor were any changes in TRAIL-R1 or -R4 mRNA or protein detected (data not shown). Thus, TRAIL expression can be detected on Mφ within 2 h after IFN-γ stimulation, paired with a concomitant loss in cell surface expression of the cognate death-inducing TRAIL-R2. Similar results examining TRAIL and TRAIL receptor expression on IFN-α–stimulated Mφ were also detected (data not shown). The loss of TRAIL-R2 expression suggested that peripheral blood Mφ stimulated with IFN-γ would be resistant to TRAIL-mediated death. Thus, Mφ were cultured in the absence or presence of GM-CSF or IFN-γ and then examined for sensitivity to LZ-TRAIL. Mφ treated with GM-CSF displayed increased sensitivity to TRAIL-induced death compared with untreated Mφ; in contrast, IFN-γ treatment significantly decreased TRAIL-induced Mφ death . Similar results were obtained with IFN-α–stimulated Mφ (data not shown). No significant changes in Mφ sensitivity to TRAIL were seen with the other cytokines tested (IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, and IL-15; data not shown) compared with untreated Mφ. These results suggest that Mφ stimulated with IFN-γ minimize TRAIL-mediated suicide or fratricide with the downregulation of TRAIL-R2 surface levels. However, it remains possible that additional mechanisms may contribute to the protection of the Mφ from TRAIL-induced death. The rendering of Mφ resistant to TRAIL-mediated apoptosis after stimulation with IFN-γ suggested that the tumor cell targets used in Fig. 2 could be affected in a similar fashion upon culture with IFN-γ. OVCAR3 tumor cells were incubated in the absence or presence of IFN-γ for 12 h, and then tested for sensitivity to LZ-TRAIL or IFN-γ–stimulated Mφ. In contrast to the Mφ, the sensitivity of OVCAR3 tumor cells was not altered after incubation with IFN-γ . Similar results were seen with other tumor cells (WM 793 and PC-3; data not shown). These results imply that not all cell types respond to IFN-γ by gaining resistance to TRAIL-induced death as observed with the Mφ. Mφ not only influence the activities of other immune and nonimmune cells in the body, but also function as effector cells under a variety of conditions ( 1 ). Activated Mφ display potent tumoricidal activity against several different tumor cell types ( 7 , 47 – 49 ). The results presented here demonstrate that one of the mechanisms by which Mφ kill tumor cells is through expression of TRAIL. Mφ stimulation with either IFN-γ or IFN-α resulted in the rapid expression of TRAIL on the cell surface, but not FasL or TNF. Because TRAIL mediates apoptosis in a high percentage (approximately two thirds) of hematopoietic and nonhematopoietic cell types ( 30 , 36 , 38 , 42 ), Mφ have the potential to mediate apoptosis of a broad range of tumor cell types via TRAIL. Expression of TRAIL appeared to be specific to the IFNs, as Mφ stimulation with either IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, or GM-CSF resulted in no detectable TRAIL expression (data not shown). Interestingly, a concomitant loss of TRAIL-R2 (and to a lesser extent TRAIL-R3) expression was detected upon IFN stimulation, rendering the Mφ resistant to TRAIL-mediated death. To our knowledge, this is the first demonstration of inducible TRAIL expression on a particular human peripheral blood cell population, as well as modulation of the death-inducing TRAIL-R2 by a proinflammatory cytokine on the same cell population. Although the expression of TRAIL on the IFN-stimulated Mφ is critical for the tumoricidal activity in our assay system, the sensitivity of the tumor cell to TRAIL-induced apoptosis is also an essential component of this phenomenon, as demonstrated by the fact that the tumor cell lines and normal cells that were resistant to TRAIL-mediated apoptosis were also resistant to TRAIL-expressing Mφ. The identification of two TRAIL receptors with death- inducing ability and two without led to the initial hypothesis that the expression of TRAIL-R3 and/or -R4 conferred resistance to TRAIL-induced death ( 32 , 33 , 37 ). However, it is important to note that this hypothesis was formulated from reports examining the distribution of TRAIL receptor mRNA in several normal tissues and tumor cell lines and from experiments where TRAIL-R3 or -R4 was overexpressed in transfected cells. Most of the tumors used in this study express TRAIL-R3 and/or -R4 ( 38 , 42 ). When the OVCAR3, WM793, and PC3 tumor cells were cultured with IFN-γ before incubation with LZ-TRAIL or IFN-stimulated Mφ, the TRAIL receptor levels remained unchanged, and no significant change in the level of TRAIL sensitivity was observed (data not shown). Thus, the differences in sensitivity of the tumor cells to the TRAIL expressed on the Mφ or the recombinant TRAIL added in solution are probably regulated by a variety of molecular mechanisms, both inside the cell and at the surface. While our results focused on the tumoricidal activity of TRAIL-expressing Mφ, previous reports have shown these cells can also produce cytotoxic inorganic oxidants, such as NO ( 10 , 11 ). A role for NO in tumor cell killing has been documented for both human and mouse activated macrophages ( 4 , 44 , 50 ), where the toxicity of NO is mediated via mitochondrial damage, inhibition of DNA synthesis, and disruption of the tricarboxylic acid cycle, ultimately resulting in apoptosis ( 51 , 52 ). Although murine macrophages release high levels of NO after either LPS or IFN-γ stimulation, studies with human peripheral blood Mφ have reported contradictory findings ( 10 ). In some reports, Mφ stimulated with either LPS or IFN-γ (or in combination) failed to release significant levels of NO ( 12 – 14 , 44 , 53 ), whereas others have reported that IFN-α stimulation results in a slight increase in NO production ( 54 ). In our studies, addition of the NO inhibitor L-NMMA to the cytotoxicity assays did not decrease the ability of the IFN-γ– and IFN-α–stimulated Mφ to kill the tumor cell targets. Moreover, analysis of the culture supernatants for nitrites revealed no increase after 12 h stimulation with GM-CSF, IFN-γ, or IFN-α (data not shown). These observations, coupled with the fact that TRAIL-R2:Fc completely inhibited the tumoricidal activity of the Mφ to background (unstimulated Mφ) levels, imply that TRAIL is the primary mediator of the tumoricidal activity after IFN stimulation. Coexpression of TRAIL and TNF, and perhaps other unidentified death-inducing molecules, would theoretically increase both the cytolytic potential of the Mφ and the range of different tumor targets susceptible to Mφ-mediated death. The importance of IFN in the management of spontaneously arising tumors was recently demonstrated in vivo using mice that lack sensitivity to IFN-γ ( 55 ). Compared with wild-type mice, the IFN-γ–insensitive mice develop tumors more rapidly and with greater frequency when challenged with a chemical carcinogen. Part of this “IFN effect” is via the interaction of the IFN with the tumor cells by enhancing the tumor cell tumorigenicity through heightened MHC class I expression. IFN-γ may also enhance an innate antitumor mechanism through the induction of TRAIL on cells of the Mφ lineage ( 55 ). Although our data suggest that Mφ would confer this antitumor activity, further studies are required to determine if other cell types, such as NK cells, neutrophils, and dendritic cells, are also able to express TRAIL after IFN stimulation ( 56 ). Finally, in addition to a role in tumoricidal activity, these data suggest that Mφ TRAIL-expression may contribute to other physiologic and pathologic situations, such as the AICD of T cells during HIV infection. Recently, Katsikis et al. ( 57 , 58 ) have demonstrated that activation-induced peripheral blood T cell apoptosis in HIV-infected individuals was Fas independent, and a potential role for TRAIL in this phenomenon was identified. It was observed that a blocking mAb to TRAIL could inhibit the AICD of T cells in a mixed population of HIV + PBMCs ( 58 ). However, it was unclear which PBMC subset was expressing TRAIL and responsible for death of the T cells. Thus, the results presented here may provide an explanation for these experimental observations, as well as a basis for examining other activities mediated by activated Mφ. | Study | biomedical | en | 0.999998 |
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